Protein binding domains stabilizing functional conformational states of gpcrs and uses thereof

ABSTRACT

The present invention relates to the field of GPCR structure biology and signaling. In particular, the present invention relates to protein binding domains directed against or capable of specifically binding to a functional conformational state of a G-protein-coupled receptor (GPCR). More specifically, the present invention provides protein binding domains that are capable of increasing the stability of a functional conformational state of a GPCR, in particular, increasing the stability of a GPCR in its active conformational state. The protein binding domains of the present invention can be used as a tool for the structural and functional characterization of G-protein-coupled receptors bound to various natural and synthetic ligands, as well as for screening and drug discovery efforts targeting GPCRs. Moreover, the invention also encompasses the diagnostic, prognostic and therapeutic usefulness of these protein binding domains for GPCR-related diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Serial No.of U.S. patent application Ser. No. 16/045,879, filed Jul. 26, 2018,pending, which is a continuation of U.S. patent application Ser. No.15/816,505, filed Nov. 17, 2017, now U.S. Pat. No. 10,078,088, which isa continuation of U.S. patent application Ser. No. 15/409,285, filedJan. 18, 2017, now U.S. Pat. No. 9,863,959, which is a continuation ofU.S. patent application Ser. No. 15/236,398, filed Aug. 13, 2016, nowU.S. Pat. No. 9,689,872, issued Jun. 27, 2017, which is a continuationof U.S. patent application Ser. No. 13/810,652, filed Mar. 29, 2013, nowU.S. Pat. No. 9,453,065, issued Sep. 27, 2016, which is a national phaseentry under 35 U.S.C. § 371 of PCT International Patent ApplicationPCT/EP2011/062287, filed Jul. 18, 2011, designating the United States ofAmerica and published in English as International Patent Publication WO2012/007593 A1 on Jan. 19, 2012, which claims the benefit under Article8 of the Patent Cooperation Treaty and under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application Ser. No. 61/399,781, filed Jul. 16, 2010,and under Article 8 of the Patent Cooperation Treaty to United KingdomPatent Application Serial No. 1014715.5, filed Sep. 6, 2010.

GOVERNMENT RIGHTS

This invention was made with Government support under Contract No.NS028471 awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

STATEMENT ACCORDING TO 37 C.F.R. § 1.821(C) OR (E)—SEQUENCE LISTINGSUBMITTED AS ASCII TEXT FILE

Pursuant to 37 C.F.R. § 1.821(c) or (e), a file containing an ASCII textversion of the Sequence Listing has been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.

TECHNICAL FIELD

The disclosure relates to the field of GPCR structure biology andsignaling. In particular, the disclosure relates to protein bindingdomains directed against or capable of specifically binding to afunctional conformational state of a G protein-coupled receptor (GPCR).More specifically, the disclosure provides protein binding domains thatare capable of increasing the stability of a functional conformationalstate of a GPCR, in particular, increasing the stability of a GPCR inits active conformational state. The protein binding domains hereof canbe used as a tool for the structural and functional characterization ofG-protein-coupled receptors bound to various natural and syntheticligands, as well as for screening and drug discovery efforts targetingGPCRs. Moreover, the invention also encompasses the diagnostic,prognostic and therapeutic usefulness of these protein binding domainsfor GPCR-related diseases.

BACKGROUND

G protein-coupled receptors (GPCRs) are the largest family of membraneproteins in the human genome. They play essential roles in physiologicresponses to a diverse set of ligands, such as biogenic amines, aminoacids, peptides, proteins, prostanoids, phospholipids, fatty acids,nucleosides, nucleotides, Ca²⁺ ions, odorants, bitter and sweettastants, pheromones and protons (Heilker et al. 2009). GPCRs aretherapeutic targets for a broad range of diseases. GPCRs arecharacterized by seven transmembrane domains with an extracellular aminoterminus and an intracellular carboxyl terminus, and are also calledseven transmembrane or heptahelical receptors (Rosenbaum et al. 2009).Rhodopsin, a GPCR that is highly specialized for the efficient detectionof light, has been the paradigm for GPCR signaling and structuralbiology due to its biochemical stability and natural abundance in bovineretina (Hofmann et al. 2009). In contrast, many GPCRs exhibit complexfunctional behavior by modulating the activity of multiple G proteinisoforms, as well as G protein independent signaling pathways (e.g.,β-arrestin). In some cases, a GPCR may exhibit basal activity toward aspecific signaling pathway, even in the absence of a ligand. Orthostericligands that act on a GPCR can have a spectrum of effects on downstreamsignaling pathways. Full agonists maximally activate the receptor.Partial agonists elicit a submaximal stimulation, even at saturatingconcentrations. Inverse agonists inhibit basal activity, while neutralantagonists have no effect on basal activity, but competitively blockbinding of other ligands.

The complex behavior of GPCRs for hormones and neurotransmitters can beattributed to their structural plasticity (Kobilka and Deupi 2007).Evidence from functional and biophysical studies shows that GPCRs canexist in multiple functionally distinct conformational states (Kobilkaand Deupi 2007). While this structural plasticity and dynamic behavioris essential for normal function, it contributes to their biochemicalinstability and difficulty in obtaining high-resolution crystalstructures. To date, crystal structures have been reported for the humanβ₂AR (Rasmussen et al. 2007; Rosenbaum et al. 2007; Cherezov et al.2007; Hanson et al. 2008), the avian β₁AR (Warne et al. 2008), and humanA2 adenosine receptor (Jaakola et al. 2008). While rhodopsin can becrystallized from unmodified protein isolated from native tissue, theseother GPCRs required expression in recombinant systems, stabilization ofan inactive state by an inverse agonist and biochemical modifications tostabilize the receptor protein. The first crystal structure of the β₂ARwas stabilized by a selective Fab (Rasmussen et al. 2007). Subsequentstructures of the β₂AR and the A2 adenosine receptor were obtained withthe aid of protein engineering: the insertion of T4Lysozyme into thethird intracellular loop as originally described for the β₂AR (Rosenbaumet al. 2007). Finally, crystals of the avian β₁AR were grown fromprotein engineered with amino and carboxyl terminal truncations anddeletion of the third intracellular loop, as well as six amino acidsubstitutions that enhanced thermostability of the purified protein(Warne et al. 2008).

Obtaining structures of an active state of a GPCR is more difficultbecause this state is relatively unstable. Fluorescence lifetime studiesshow that the β₂AR is structurally heterogeneous in the presence ofsaturating concentrations of a full agonist (Ghanouni et al. 2001). Thisstructural heterogeneity is incompatible with the formation of crystals.Stabilization of the active state of the β₂AR requires the presence ofits cognate G protein Gs, the stimulatory protein for adenylyl cyclase(Yao et al. 2009). To date, the only active state structure of GPCR isthat of opsin, the ligand free form of rhodopsin (Park et al. 2008).These crystals were grown at acidic pH (5.5) where opsin has been shownto be structurally similar to light-activated rhodopsin (metarhodopsinII) at physiologic pH by FTIR spectroscopy. While the β₂AR also exhibitshigher basal activity at reduced pH, it is biochemically unstable(Ghanouni et al. 2000).

Unraveling the structures of different functional conformational statesof GPCRs in complex with various natural and synthetic ligands andproteins is valuable, both for understanding the mechanisms of GPCRsignal transduction as well as for structure-based drug discoveryefforts. The development of new straightforward tools forhigh-resolution structure analysis of individual conformers of GPCRs is,therefore, needed.

BRIEF SUMMARY

A first aspect hereof relates to a protein binding domain capable ofspecifically binding to a functional conformational state of a GPCR.

In one embodiment, the protein binding domain is capable of stabilizinga functional conformational state of a GPCR upon binding. Preferably,the protein binding domain is capable of inducing a functionalconformational state in a GPCR upon binding.

In another embodiment, the functional conformational state of a GPCR isselected from the group consisting of a basal conformational state, oran active conformational state or an inactive conformational state.Preferably, the functional conformational state of a GPCR is an activeconformational state.

According to another embodiment, the protein binding domain is capableof specifically binding to an agonist-bound GPCR and/or enhances theaffinity of a GPCR for an agonist.

According to another embodiment, the protein binding domain is capableof increasing the thermostability of a functional conformational stateof a GPCR upon binding.

In a specific embodiment, the protein binding domain is capable ofspecifically binding to a conformational epitope of the functionalconformational state of a GPCR. Preferably, the conformational epitopeis an intracellular epitope. More preferably, the conformational epitopeis comprised in a binding site for a downstream signaling protein. Mostpreferably, the conformational epitope is comprised in the G proteinbinding site.

Preferably, the protein binding domain hereof comprises an amino acidsequence that comprises four framework regions and threecomplementarity-determining regions, or any suitable fragment thereof.More preferably, the protein binding domain is derived from a camelidantibody. Most preferably, the protein binding domain comprises ananobody sequence, or any suitable fragment thereof. For example, thenanobody comprises a sequence selected from the group consisting of SEQID NOS:1-29, or any suitable fragment thereof.

According to another embodiment, the GPCR is a mammalian protein, or aplant protein, or a microbial protein, or a viral protein, or an insectprotein. The mammalian protein can be a human protein. In particular,the GPCR is chosen from the group comprising a GPCR of the Glutamatefamily of GPCRs, a GPCR of the Rhodopsin family of GPCRs, a GPCR of theAdhesion family of GPCRs, a GPCR of the Frizzled/Taste2 family of GPCRs,and a GPCR of the Secretin family of GPCRs. More specifically, the GPCRis an adrenergic receptor, such as an α-adrenergic receptor or aβ-adrenergic receptor, or wherein the GPCR is a muscarinic receptor,such as an M₁-muscarinic receptor or an M₂-muscarinic receptor, or anM₃-muscarinic receptor, or an M₄-muscarinic receptor, or anM₅-muscarinic receptor, or wherein the GPCR is an angiotensin receptor,such as an angiotensin II type 1 receptor or an angiotensin II type 2receptor.

A second aspect hereof relates to a complex comprising: (i) a proteinbinding domain hereof, (ii) a GPCR in a functional conformational state,and (iii) optionally, a receptor ligand. The receptor ligand can bechosen from the group comprising a small molecule, a protein, a peptide,a protein scaffold, a nucleic acid, an ion, a carbohydrate or anantibody, or any suitable fragment thereof. The complex can be in asolubilized form or immobilized to a solid support. In particular, thecomplex is crystalline. The invention further encompasses a crystallineform of a complex comprising (i) a protein binding domain, (ii) a GPCRin a functional conformational state, and (iii) optionally, a receptorligand, wherein the crystalline form is obtained by the use of a proteinbinding domain hereof.

A third aspect hereof relates to a cellular composition comprising aprotein binding domain hereof and/or a complex hereof. Preferably, theprotein binding domain comprised in the cellular composition is capableof stabilizing and/or inducing a functional conformational state of aGPCR upon binding of the protein binding domain.

A fourth aspect hereof relates to the use of a protein binding domainhereof or a complex hereof or a cellular composition hereof to stabilizeand/or induce a functional conformational state of a GPCR.

According to one embodiment, the protein binding domain or the complexor the cellular composition can be used to crystallize and/or to solvethe structure of a GPCR in a functional conformational state.

Also encompassed is a method of determining a crystal structure of aGPCR in a functional conformational state, the method comprising thesteps of:

-   -   (i) providing a protein binding domain hereof, a target GPCR,        and optionally a receptor ligand,    -   (ii) forming a complex of the protein binding domain, the GPCR,        and optionally the receptor ligand, and    -   (iii) crystallizing the complex of step (ii) to form a crystal,

wherein the crystal structure is determined of a GPCR in a functionalconformational state.

The above method of determining a crystal structure of a GPCR mayfurther comprise the step of obtaining the atomic coordinates from thecrystal.

According to another embodiment, the protein binding domain or thecomplex or the cellular composition can be used to capture a GPCR in afunctional conformational state, optionally with a receptor ligand orwith one or more downstream signaling proteins.

Also encompassed is a method of capturing a GPCR in a functionalconformational state, the method comprising the steps of:

-   -   (i) providing a protein binding domain hereof and a target GPCR,        and    -   (ii) forming a complex of the protein binding domain and the        GPCR,

wherein a GPCR is captured in a functional conformational state.

Further, also encompassed is a method of capturing a GPCR in afunctional conformational state, the method comprising the steps of:

-   -   (i) applying a solution containing a GPCR in a plurality of        conformational states to a solid support possessing an        immobilized protein binding domain hereof,    -   (ii) forming a complex of the protein binding domain and the        GPCR, and    -   (iii) removing weakly bound or unbound molecules,

wherein a GPCR is captured in a functional conformational state.

The above methods of capturing a GPCR in a functional conformationalstate may comprise the step of purifying the complex.

According to another embodiment, also disclosed is the use of theprotein binding domain, or the complex, or the cellular composition,hereof, in screening and/or identification programs forconformation-specific (drug) compounds or ligands of a GPCR.

Also encompassed is a method of identifying compounds capable of bindingto a functional conformational state of a GPCR, the method comprisingthe steps of:

-   -   (i) Providing a GPCR and a protein binding domain hereof,    -   (ii) Providing a test compound, and    -   (iii) Evaluating whether the test compound binds to the        functional conformational state of the GPCR, and    -   (iv) Selecting a compound that binds to the functional        conformational state of the GPCR.

Preferably, the above-described method for identifying compounds furthercomprises the step of forming a complex comprising the protein bindingdomain and the GPCR in a functional conformational state, hereof. Thecomplex may further comprise a receptor ligand, which can be chosen fromthe group comprising a small molecule, a protein, a peptide, a proteinscaffold, a nucleic acid, an ion, a carbohydrate or an antibody, or anysuitable fragment thereof. Preferably, the receptor ligand is a fullagonist, or a partial agonist, or an inverse agonist, or an antagonist.Preferably, the protein binding domain and/or the complex are providedin essentially purified form. Alternatively, the protein binding domainand/or the complex are provided in a solubilized form. Alternatively,the protein binding domain and/or the complex is immobilized to a solidsupport. Alternatively, the protein binding domain and/or the complex isprovided in a cellular composition.

According to another embodiment, the test compound used in theabove-described method for identifying compounds is selected from thegroup comprising a polypeptide, a peptide, a small molecule, a naturalproduct, a peptidomimetic, a nucleic acid, a lipid, a lipopeptide, acarbohydrate, an antibody or any fragment derived thereof, such as Fab,Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (dsFv) and fragments comprising either a VL or VHdomain, a heavy chain antibody (hcAb), a single domain antibody (sdAb),a minibody, the variable domain derived from camelid heavy chainantibodies (VHH or nanobody), the variable domain of the new antigenreceptors derived from shark antibodies (VNAR), a protein scaffoldincluding an alphabody, protein A, protein G, designed ankyrin-repeatdomains (DARPins), fibronectin type III repeats, anticalins, knottins,or engineered CH2 domains (nanoantibodies).

Preferably, the test compounds are labeled. Further, a library of testcompounds may be used. Further, the above-described method foridentifying compounds may be a high-throughput screening method.

According to another specific embodiment, the protein binding domain orthe complex or the cellular composition, all hereof, can be used todiagnose or prognose a GPCR-related disease, such as cancer, autoimmunedisease, infectious disease, neurological disease, or cardiovasculardisease.

A fifth aspect hereof relates to a pharmaceutical composition comprisinga therapeutically effective amount of a protein binding domain hereofand at least one of a pharmaceutically acceptable carrier, adjuvant ordiluent.

A sixth aspect hereof relates to the use of a protein binding domainhereof or a pharmaceutical composition hereof to modulate GPCR signalingactivity, more specifically, to block G-protein-mediated signaling.

The protein binding domain or the pharmaceutical composition hereof mayalso be used in the treatment of a GPCR-related disease, such as cancer,autoimmune disease, infectious disease, neurological disease, orcardiovascular disease.

A seventh aspect hereof relates to a kit comprising a protein bindingdomain hereof or a complex hereof or a cellular composition hereof.

Other applications and uses of the amino acid sequences and polypeptideshereof will become clear to the skilled person from the furtherdisclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. β₂AR-specific nanobodies bind and stabilize an active state ofthe receptor:

Panel a: Representative trace of size-exclusion chromatography (SEC) forNb80. Purified β₂AR (20 μM) bound to an agonist (β₂AR-agonist) wasincubated with and without 40 μM Nb80 (black and blue, respectively) fortwo hours at room temperature prior to analyzing by FPLC. In thepresence of Nb80, the β₂AR-agonist elution peak increases in UVabsorbance (280 nm) and elutes at an earlier volume than β₂AR-agonistalone, with a simultaneous decrease in the Nb80 elution peak (green),suggesting β₂AR-agonist-Nb80 complex formation. Incubation of β₂AR (20μM) bound to an inverse agonist with Nb80 (red) resulted in a smallershift and smaller increase in UV absorbance when compared to theβ₂AR-agonist-Nb80 complex.

Panel b: Dose-response competition binding experiments on Sf9 insectcell membranes expressing β₂AR. Seven nanobodies that bound β₂AR by SECwere individually incubated for 90 minutes at room temperature withβ₂AR-expressing membranes. All seven nanobodies increased the affinityof β₂AR for (-)-isoproterenol (Table 3). Nb80 (blue) was selected as thelead nanobody. Data represent the mean±s.e. of two independentexperiments performed in triplicate.

Panel c: A fluorescence-based functional assay using monobromobimane-(mBBR-) labeled purified receptor shows that 1 μM Nb80 (blue) stabilizesa more active state of the β₂AR (bound to the full agonist(-)-isoproterenol) when compared with receptor in the absence of Nb80(black). The active state is characterized by a quenching of mBBrfluorescence and a redshift in mBBr fluorescence (Yao et al. 2009).

FIG. 2. Representative dot blots showing specificity of nanobodies tothe tertiary structure of the β₂AR:

Panel a: Equal amounts of native or SDS-denatured purified β₂AR bound toan agonist (top and middle, respectively), or native β₂AR bound to aninverse agonist (bottom) were spotted in triplicate on nitrocellulosestrips. The strips were blocked with 5% nonfat dry milk in PBS (pH 7.4)with 0.05% TWEEN®-20 and then incubated with 1 mg/ml of indicatednanobodies diluted in blocking buffer. Binding of nanobodies wasdetected by an anti-histidine (a-6His) primary mouse antibody, followedby incubation with goat-anti-mouse IR-800-labeled secondary antibody. M1antibody, which recognizes the linear FLAG epitope, was labeled withAlexa-688 and directly detected β₂AR. Dot blots were scanned and imagedusing the Odyssey Infrared Imaging System (Li-cor Biosciences). Blotsdetecting nanobodies were processed separately from blots detecting β₂ARby M1 since two different channels (800 nm versus 700 nm, respectively)were used for imaging; therefore, blots cannot be directly compared andquantified (i.e., comparison of binding of nanobodies versus M1 bindingare only qualitative in nature).

Panel b: Representative dot blots showing nanobodies with reducedbinding to natively folded β₂AR.

FIGS. 3A and 3B. Selective binding of nanobodies to the active state ofthe receptor: Purified β₂AR (20 μM) bound to an agonist was incubatedwith and without 40 μM nanobodies (black and blue, respectively) for twohours at room temperature prior to analyzing by size exclusionchromatography. Samples of β₂AR (20 μM) bound to an inverse agonist inthe presence of nanobodies (red) were also analyzed. In the presence ofseveral nanobodies (Nb72, Nb65, Nb71, Nb69, Nb67 and Nb84), theβ₂AR-agonist elution peak increases in UV absorbance (280 nm) and elutesat an earlier volume (black line) than β₂AR-agonist alone (blue), with asimultaneous decrease in the Nb80 elution peak (green), suggestingβ₂AR-agonist-Nb80 complex formation. The formation of a β₂AR-inverseagonist-Nb80 complex is not observed (red line).

FIG. 4. Selective binding of nanobodies to the active state of thereceptor: Purified β₂AR (20 μM) bound to an agonist was incubated withand without 40 μM nanobodies (black and blue, respectively) for twohours at room temperature prior to analyzing by size exclusionchromatography.

FIGS. 5A-5F. Fluorescence emission spectra showing nanobody-inducedconformational changes of monobromobimane-labeled β₂AR: Nanobodies thatincrease agonist binding affinity for the β₂AR stabilize an active stateof the receptor. A fluorescence-based functional assay usingmonobromobimane- (mBBr-) labeled purified receptor shows that 1 μM ofnanobodies 65, 67, 69, 71, 72 and 84 (red) stabilize a more active stateof the β₂AR (bound to the full agonist isoproterenol) when compared withreceptor in the absence of nanobodies (black). This active state ischaracterized by a quenching of mBBr fluorescence and a redshift in mBBrfluorescence (Yao et al. 2009).

FIG. 6. Effect of Nb80 on β₂AR structure and function:

Panel A: The cartoon illustrates the movement of the environmentallysensitive bimane probe attached to Cys265^(6,27) in the cytoplasmic endof TM6 from a more buried, hydrophobic environment to a more polar,solvent-exposed position during receptor activation that results in adecrease in the observed fluorescence in FIG. 6, Panels B and C.

Panels B and C: Fluorescence emission spectra showing ligand-inducedconformational changes of monobromobimane-labeled β₂AR reconstitutedinto high density lipoprotein particles (mBB-β₂AR/HDL) in the absence(black solid line) or presence of full agonist isoproterenol (ISO, greenwide dashed line), inverse agonist ICI-118,551 (ICI, black dashed line),G_(s) heterotrimer (red solid line), nanobody-80 (Nb80, blue solidlines), and combinations of G_(s) with ISO (red wide dashed line), Nb80with ISO (blue wide dashed line), and Nb80 with ICI (blue dashed line).

Panels D through F: Ligand binding curves for ISO competing against[³H]-dihydroalprenolol ([³H]-DHA) for Panel D, β₂AR/HDL reconstitutedwith G_(s) heterotrimer in the absence or presence GTPγS, Panel E,β₂AR/HDL in the absence and presence of Nb80, and Panel F, β₂AR-T4L/HDLin the absence and presence of Nb80. Error bars represent standarderrors.

FIG. 7. Packing of the agonist-β₂AR-T4L-Nb80 complex in crystals formedin lipidic cubic phase: Three different views of the structure of β₂ARindicated in orange, Nb80 in blue, and agonist in green. T4 lysozyme(T4L) could not be modeled due to poor electron density; its likelyposition is indicated by the light blue circle with black dashed linesconnected to the intracellular ends of TM5 and TM6 where it is fused inthe β₂AR-T4L construct. PyMOL (http://www.pymol.org) was used for thepreparation of all figures.

FIG. 8. Comparison of the inverse agonist and agonist-Nb80 stabilizedcrystal structures of the β₂AR: The structure of inverse agonistcarazolol-bound β₂AR-T4L (β₂AR-Cz) is shown in blue with the carazololin yellow. The structure of agonist bound and Nb80 stabilized β₂AR-T4L(β₂AR-Nb80) is shown in orange with agonist in green. These twostructures were aligned using Pymol align function.

Panel A: Side view of the superimposed structures showing significantstructural changes in the intracellular and G protein facing part of thereceptors.

Panel B: Side view following 90 degrees rotation on the vertical axis.

Panel C: Comparison of the extracellular ligand binding domains showingmodest structural changes.

FIG. 9. Nb80 stabilized intracellular domain compared to inactive β₂ARand opsin structures:

Panel A: Side view of β₂AR (orange) with CDRs of Nb80 in light blue(CDR1) and blue (CDR3) interacting with the receptor.

Panel B: Closer view focusing on CDRs 1 and 3 entering the β₂AR. Sidechains in TM3, 5, 6, and 7 within 4 Å of the CDRs are shown. The largerCDR3 penetrates 13 Å into the receptor.

Panel C: Interaction of CDR1 and CDR3 viewed from the intracellularside.

Panel D: The agonist bound and Nb80 stabilized β₂AR-T4L (β₂AR-Nb80) issuperimposed with the carazolol-bound inactive structure of β₂AR-T4L(β₂AR-Cz). The ionic lock interaction between Asp3.49 and Arg3.50 of theDRY motif in TM3 is broken in the β₂AR-Nb80 structure. The intracellularend of TM6 is moved outward and away from the core of the receptor. Thearrow indicates a 11.4 Å change in distance between the α-carbon ofGlu6.30 in the structures of β₂AR-Cz and β₂AR-Nb80. The intracellularends of TM3 and TM7 move toward the core by 4 Å and 2.5 Å, respectively,while TM5 moves outward by 6 Å.

Panel E: The β₂AR-Nb80 structure superimposed with the structure ofopsin crystallized with the C-terminal peptide of G_(t) (transducin).

FIG. 10. Nb80 stabilized intracellular domain of β₂AR compared to opsinstructures:

Panel A: Interactions between the β₂AR and Nb80.

Panel B: Interactions between opsin and the carboxyl terminal peptide oftransducin.

FIG. 11. Rearrangement of transmembrane segment packing interactionsupon agonist binding:

Panel A: Packing interactions that stabilize the inactive state areobserved between Pro211 in TM5, Ile121 in TM3, Phe282 in TM6 and Asn316in TM5.

Panel B: The inward movement of TM5 upon agonist binding disrupts thepacking of Ile121 and Pro211 resulting in a rearrangement ofinteractions between Ile121 and Phe282. These changes contribute to arotation and outward movement of TM6 and an inward movement of TM7.

FIG. 12. Amino acid sequences of the different nanobodies raised againstβ₂AR: Sequences have been aligned using standard software tools. CDRshave been defined according to IMGT numbering (Lefranc et al. 2003).

FIG. 13. Effect of Nb80 on the thermal stability of the β₂AR receptor:Comparison of the melting curves of detergent-solubilized (DDM)agonist-bound (isoproterenol) β₂AR in the presence and absence of Nb80.The apparent melting temperature for β₂AR without Nb80 is 12.0° C. Theapparent melting temperature for β₂AR with Nb80 is 24° C.

FIGS. 14A and 14B. Effect of Nb80 on the temperature-induced aggregationof the β₂AR receptor:

FIG. 14A: Detergent-solubilized (DDM) β₂AR was heated for ten minutes at50° C. in the presence of Nb80 or isoproterenol and the aggregation ofthe receptor was analyzed by SEC.

FIG. 14B: Temperature dependence of the isoproterenol-bound receptor inthe absence of Nb80.

FIG. 15. Nb80 has little effect on β₂AR binding to the inverse agonistICI-118,551: β₂AR (Panel A) or β₂AR-T4L (Panel B) was reconstituted intoHDL particles and agonist competition binding experiments were performedin the absence or presence of Nb80. Ligand binding curves for theinverse agonist ICI-118551 competing against [³H]-dihydroalprenolol([3H]-DHA) for a, β₂AR/HDL in the absence and presence of Nb80, and b,β₂AR-T4L/HDL in the absence and presence of Nb80.

FIGS. 16A and 16B. Nb80 increases β₂AR affinity for agonists but not forantagonists: Competitive ligand binding experiments were performed oncommercial insect cell-derived membranes containing full-length β₂AR inthe absence or presence of Nb80. Dose-dependent radio-liganddisplacement curves in presence of Nb80 and an irrelevant Nanobody (IrrNb) for two representative agonists (isoproterenol, procaterol) and tworepresentative antagonists (ICI-118,551 and carvedilol).

FIG. 17. Sequence alignment of human β₁AR and human β₂AR: Amino acids ofthe β₂-adrenoreceptor that interact with Nb80 in the β₂AR-Nb80 interfaceare underlined.

FIGS. 18A-18D. Nb80 selectively binds the active conformation of thehuman β₁AR receptor: Ligand binding curves for agonists and inverseagonists competing against [³H]-dihydroalprenolol ([³H]-DHA).

FIG. 18A: Agonist Isoproterenol (ISO) binding to β₂AR in the presenceand absence of Nb80.

FIG. 18B: Inverse agonist ICI-118,551 (ICI) binding to β₂AR in thepresence and absence of Nb80.

FIG. 18C: Agonist Isoproterenol (ISO) binding to β₁AR in the presenceand absence of Nb80.

FIG. 18D: Inverse agonist CGP20712A (CPG) binding to β₁AR in thepresence and absence of Nb80.

DETAILED DESCRIPTION Definitions

The disclosure is described with respect to particular embodiments andwith reference to certain drawings; the invention is not limitedthereto, but only by the claims. Any reference signs in the claims shallnot be construed as limiting the scope. The drawings described are onlyschematic and are non-limiting. In the drawings, the size of some of theelements may be exaggerated and not drawn on scale for illustrativepurposes. Where the term “comprising” is used in the present descriptionand claims, it does not exclude other elements or steps. Where anindefinite or definite article is used when referring to a singularnoun, e.g., “a,” “an,” or “the,” this includes a plural of that noununless something else is specifically stated. Furthermore, the terms“first,” “second,” “third,” and the like in the description and in theclaims, are used for distinguishing between similar elements and notnecessarily for describing a sequential or chronological order. It is tobe understood that the terms so used are interchangeable underappropriate circumstances and that the embodiments hereof describedherein are capable of operation in other sequences than described orillustrated herein.

Unless otherwise defined herein, scientific and technical terms andphrases used in connection with the disclosure shall have the meaningsthat are commonly understood by those of ordinary skill in the art.Generally, nomenclatures used in connection with, and techniques ofmolecular and cellular biology, genetics and protein and nucleic acidchemistry and hybridization described herein, are those well known andcommonly used in the art. The methods and techniques hereof aregenerally performed according to conventional methods well known in theart and as described in various general and more specific referencesthat are cited and discussed throughout the present specification unlessotherwise indicated. See, for example, Sambrook et al., MolecularCloning: A Laboratory Manual, 2d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989); Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates (1992, andSupplements to 2002).

The term “protein binding domain” refers generally to any non-naturallyoccurring molecule or part thereof that is able to bind to a protein orpeptide using specific intermolecular interactions. A variety ofmolecules can function as protein binding domains, including, but notlimited to, proteinaceous molecules (protein, peptide, protein-like orprotein containing), nucleic acid molecules (nucleic acid, nucleicacid-like, nucleic acid containing), and carbohydrate molecules(carbohydrate, carbohydrate-like, carbohydrate containing). A moredetailed description can be found further in the specification.

As used herein, the terms “polypeptide,” “protein,” and “peptide” areused interchangeably herein, and refer to a polymeric form of aminoacids of any length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones.

As used herein, the terms “multiprotein complex” or “protein complex” orsimply “complex” refer to a group of two or more associated polypeptidechains. Proteins in a protein complex are linked by non-covalentprotein-protein interactions. The “quaternary structure” is thestructural arrangement of the associated folded proteins in the proteincomplex. A “multimeric complex” refers to a protein complex as definedherein, which may further comprise a non-proteinaceous molecule.

As used herein, the terms “nucleic acid molecule,” “polynucleotide,”“polynucleic acid,” and “nucleic acid” are used interchangeably andrefer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three-dimensional structure, and mayperform any function, known or unknown. Non-limiting examples ofpolynucleotides include a gene, a gene fragment, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, control regions, isolated RNA ofany sequence, nucleic acid probes, and primers. The nucleic acidmolecule may be linear or circular.

As used herein, the term “ligand” or “receptor ligand” means a moleculethat specifically binds to a GPCR, either intracellularly orextracellularly. A ligand may be, without the purpose of beinglimitative, a protein, a (poly)peptide, a lipid, a small molecule, aprotein scaffold, a nucleic acid, an ion, a carbohydrate, an antibody oran antibody fragment, such as a nanobody (all as defined herein). Aligand may be a synthetic or naturally occurring. A ligand also includesa “native ligand,” which is a ligand that is an endogenous, naturalligand for a native GPCR. A “modulator” is a ligand that increases ordecreases the signaling activity of a GPCR (i.e., via an intracellularresponse) when it is in contact with, e.g., binds to, a GPCR that isexpressed in a cell. This term includes agonists, full agonists, partialagonists, inverse agonists, and antagonists, of which a more detaileddescription can be found further in the specification.

The term “conformation” or “conformational state” of a protein refersgenerally to the range of structures that a protein may adopt at anyinstant in time. One of skill in the art will recognize thatdeterminants of conformation or conformational state include a protein'sprimary structure as reflected in a protein's amino acid sequence(including modified amino acids) and the environment surrounding theprotein. The conformation or conformational state of a protein alsorelates to structural features such as protein secondary structures(e.g., α-helix, β-sheet, among others), tertiary structure (e.g., thethree-dimensional folding of a polypeptide chain), and quaternarystructure (e.g., interactions of a polypeptide chain with other proteinsubunits). Post-translational and other modifications to a polypeptidechain such as ligand binding, phosphorylation, sulfation, glycosylation,or attachments of hydrophobic groups, among others, can influence theconformation of a protein. Furthermore, environmental factors, such aspH, salt concentration, ionic strength, and osmolality of thesurrounding solution, and interaction with other proteins andco-factors, among others, can affect protein conformation. Theconformational state of a protein may be determined by either functionalassay for activity or binding to another molecule or by means ofphysical methods such as X-ray crystallography, NMR, or spin labeling,among other methods. For a general discussion of protein conformationand conformational states, one is referred to Cantor and Schimmel,Biophysical Chemistry, Part I: The Conformation of BiologicalMacromolecules, W.H. Freeman and Company, 1980, and Creighton, Proteins:Structures and Molecular Properties, W.H. Freeman and Company, 1993. A“specific conformational state” is any subset of the range ofconformations or conformational states that a protein may adopt.

A “functional conformation” or a “functional conformational state,” asused herein, refers to the fact that proteins possess differentconformational states having a dynamic range of activity, in particular,ranging from no activity to maximal activity. It should be clear that “afunctional conformational state” is meant to cover any conformationalstate of a GPCR, having any activity, including no activity; and is notmeant to cover the denatured states of proteins.

As used herein, the terms “complementarity-determining region” or “CDR”within the context of antibodies refer to variable regions of either H(heavy) or L (light) chains (also abbreviated as VH and VL,respectively) and contains the amino acid sequences capable ofspecifically binding to antigenic targets. These CDR regions account forthe basic specificity of the antibody for a particular antigenicdeterminant structure. Such regions are also referred to as“hypervariable regions.” The CDRs represent non-contiguous stretches ofamino acids within the variable regions but, regardless of species, thepositional locations of these critical amino acid sequences within thevariable heavy and light chain regions have been found to have similarlocations within the amino acid sequences of the variable chains. Thevariable heavy and light chains of all canonical antibodies each havethree CDR regions, each non-contiguous with the others (termed L1, L2,L3, H1, H2, H3) for the respective light (L) and heavy (H) chains.Nanobodies, in particular, generally comprise a single amino acid chainthat can be considered to comprise four “framework sequences or regions”or FRs and three complementarity-determining regions” or CDRs. Thenanobodies have three CDR regions, each non-contiguous with the others(termed CDR1, CDR2, CDR3). The delineation of the FR and CDR sequencesis based on the IMGT unique numbering system for V-domains and V-likedomains (Lefranc et al. 2003).

An “epitope,” as used herein, refers to an antigenic determinant of apolypeptide. An epitope could comprise three amino acids in a spatialconformation, which is unique to the epitope. Generally an epitopeconsists of at least 4, 5, 6, or 7 such amino acids, and more usually,consists of at least 8, 9, or 10 such amino acids. Methods ofdetermining the spatial conformation of amino acids are known in the artand include, for example, x-ray crystallography and two-dimensionalnuclear magnetic resonance.

A “conformational epitope,” as used herein, refers to an epitopecomprising amino acids in a spatial conformation that is unique to afolded three-dimensional conformation of the polypeptide. Generally, aconformational epitope consists of amino acids that are discontinuous inthe linear sequence that come together in the folded structure of theprotein. However, a conformational epitope may also consist of a linearsequence of amino acids that adopts a conformation that is unique to afolded three-dimensional conformation of the polypeptide (and notpresent in a denatured state). In multiprotein complexes, conformationalepitopes consist of amino acids that are discontinuous in the linearsequences of one or more polypeptides that come together upon folding ofthe different folded polypeptides and their association in a uniquequaternary structure. Similarly, conformational epitopes may here alsoconsist of a linear sequence of amino acids of one or more polypeptidesthat come together and adopt a conformation that is unique to thequaternary structure.

The term “specificity,” as used herein, refers to the ability of aprotein binding domain, in particular, an immunoglobulin or animmunoglobulin fragment, such as a nanobody, to bind preferentially toone antigen versus a different antigen, and does not necessarily implyhigh affinity.

The term “affinity,” as used herein, refers to the degree to which aprotein binding domain, in particular, an immunoglobulin such as anantibody, or an immunoglobulin fragment such as a nanobody, binds to anantigen so as to shift the equilibrium of antigen and protein bindingdomain toward the presence of a complex formed by their binding. Thus,for example, where an antigen and antibody (fragment) are combined inrelatively equal concentration, an antibody (fragment) of high affinitywill bind to the available antigen so as to shift the equilibrium towardhigh concentration of the resulting complex. The dissociation constantis commonly used to describe the affinity between the protein bindingdomain and the antigenic target. Typically, the dissociation constant islower than 10⁻⁵ M. Preferably, the dissociation constant is lower than10⁻⁶ M; more preferably, lower than 10⁻⁷ M. Most preferably, thedissociation constant is lower than 10⁻⁸M.

The terms “specifically bind” and “specific binding,” as used herein,generally refers to the ability of a protein binding domain, inparticular, an immunoglobulin such as an antibody, or an immunoglobulinfragment such as a nanobody, to preferentially bind to a particularantigen that is present in a homogeneous mixture of different antigens.In certain embodiments, a specific binding interaction will discriminatebetween desirable and undesirable antigens in a sample, in someembodiments more than about ten- to 100-fold or more (e.g., more thanabout 1000- or 10,000-fold). Within the context of the spectrum ofconformational states of GPCRs, the terms particularly refer to theability of a protein binding domain (as defined herein) topreferentially recognize and/or bind to a particular conformationalstate of a GPCR as compared to another conformational state. Forexample, an active state-selective protein binding domain willpreferentially bind to a GPCR in an active conformational state and willnot, or to a lesser degree, bind to a GPCR in an inactive conformationalstate, and will thus have a higher affinity for the activeconformational state. The terms “specifically bind,” “selectively bind,”“preferentially bind,” and grammatical equivalents thereof, are usedinterchangeably herein. The terms “conformational specific” or“conformational selective” are also used interchangeably herein.

An “antigen,” as used herein, means a molecule capable of eliciting animmune response in an animal. Within the context of the spectrum ofconformational states of GPCR, the molecule comprises a conformationalepitope of a GPCR in a particular conformational state that is notformed or less accessible in another conformational state of the GPCR.

A “deletion” is defined here as a change in either amino acid ornucleotide sequence in which one or more amino acid or nucleotideresidues, respectively, are absent as compared to an amino acid sequenceor nucleotide sequence of a parental polypeptide or nucleic acid. Withinthe context of a protein, a deletion can involve deletion of about two,about five, about ten, up to about twenty, up to about thirty, or up toabout fifty or more amino acids. A protein or a fragment thereof maycontain more than one deletion. Within the context of a GPCR, a deletionmay also be a loop deletion, or an N- and/or C-terminal deletion.

An “insertion” or “addition” is that change in an amino acid ornucleotide sequence that has resulted in the addition of one or moreamino acid or nucleotide residues, respectively, as compared to an aminoacid sequence or nucleotide sequence of a parental protein. “Insertion”generally refers to addition of one or more amino acid residues withinan amino acid sequence of a polypeptide, while “addition” can be aninsertion or refer to amino acid residues added at an N- or C-terminus,or both termini. Within the context of a protein or a fragment thereof,an insertion or addition is usually of about one, about three, aboutfive, about ten, up to about twenty, up to about thirty, or up to aboutfifty or more amino acids. A protein or fragment thereof may containmore than one insertion.

A “substitution,” as used herein, results from the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively, as compared to an amino acid sequence or nucleotidesequence of a parental protein or a fragment thereof. It is understoodthat a protein or a fragment thereof may have conservative amino acidsubstitutions that have substantially no effect on the protein'sactivity. By “conservative substitutions” is intended combinations suchas gly, ala; val, ile, leu, met; asp, glu; asn, gln; ser, thr; lys, arg;cys, met; and phe, tyr, trp.

“Crystal” or “crystalline structure,” as used herein, refers to a solidmaterial, whose constituent atoms, molecules, or ions are arranged in anorderly repeating pattern extending in all three spatial dimensions. Theprocess of forming a crystalline structure from a fluid or frommaterials dissolved in the fluid is often referred to as“crystallization” or “crystallogenesis.” Protein crystals are almostalways grown in solution. The most common approach is to lower thesolubility of its component molecules gradually. Crystal growth insolution is characterized by two steps: nucleation of a microscopiccrystallite (possibly having only 100 molecules), followed by growth ofthat crystallite, ideally to a diffraction-quality crystal.

“X-ray crystallography,” as used herein, is a method of determining thearrangement of atoms within a crystal, in which a beam of X-rays strikesa crystal and diffracts into many specific directions. From the anglesand intensities of these diffracted beams, a crystallographer canproduce a three-dimensional picture of the density of electrons withinthe crystal. From this electron density, the mean positions of the atomsin the crystal can be determined, as well as their chemical bonds, theirdisorder and various other information.

The term “atomic coordinates,” as used herein, refers to a set ofthree-dimensional coordinates for atoms within a molecular structure. Inone embodiment, atomic coordinates are obtained using X-raycrystallography according to methods well known to those of ordinaryskill in the art of biophysics. Briefly described, X-ray diffractionpatterns can be obtained by diffracting X-rays off a crystal. Thediffraction data are used to calculate an electron density map of theunit cell comprising the crystal; the maps are used to establish thepositions of the atoms (i.e., the atomic coordinates) within the unitcell. Those skilled in the art understand that a set of structurecoordinates determined by X-ray crystallography contains standarderrors. In other embodiments, atomic coordinates can be obtained usingother experimental biophysical structure determination methods that caninclude electron diffraction (also known as electron crystallography)and nuclear magnetic resonance (NMR) methods. In yet other embodiments,atomic coordinates can be obtained using molecular modeling tools, whichcan be based on one or more of ab initio protein folding algorithms,energy minimization, and homology-based modeling. These techniques arewell known to persons of ordinary skill in the biophysical andbioinformatic arts.

“Solving the structure,” as used herein, refers to determining thearrangement of atoms or the atomic coordinates of a protein, and isoften done by a biophysical method, such as X-ray crystallography.

The term “compound” or “test compound” or “candidate compound” or “drugcandidate compound,” as used herein, describes any molecule, eithernaturally occurring or synthetic that is tested in an assay, such as ascreening assay or drug discovery assay. As such, these compoundscomprise organic or inorganic compounds. The compounds includepolynucleotides, lipids or hormone analogs that are characterized by lowmolecular weights. Other biopolymeric organic test compounds includesmall peptides or peptide-like molecules (peptidomimetics) comprisingfrom about two to about forty amino acids and larger polypeptidescomprising from about forty to about five hundred amino acids, such asantibodies, antibody fragments or antibody conjugates. Test compoundscan also be protein scaffolds. For high-throughput purposes, testcompound libraries may be used, such as combinatorial or randomizedlibraries that provide a sufficient range of diversity. Examplesinclude, but are not limited to, natural compound libraries, allostericcompound libraries, peptide libraries, antibody fragment libraries,synthetic compound libraries, fragment-based libraries, phage-displaylibraries, and the like. A more detailed description can be foundfurther in the specification.

As used herein, the terms “determining,” “measuring,” “assessing,”“monitoring” and “assaying” are used interchangeably and include bothquantitative and qualitative determinations.

The term “biologically active,” with respect to a GPCR, refers to a GPCRhaving a biochemical function (e.g., a binding function, a signaltransduction function, or an ability to change conformation as a resultof ligand binding) of a naturally occurring GPCR.

The terms “therapeutically effective amount,” “therapeutically effectivedose” and “effective amount,” as used herein, mean the amount needed toachieve the desired result or results.

The term “pharmaceutically acceptable,” as used herein, means a materialthat is not biologically or otherwise undesirable, i.e., the materialmay be administered to an individual along with the compound withoutcausing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

DESCRIPTION

Structural information on GPCRs will provide insight into thestructural, functional and biochemical changes involved in signaltransfer from the receptor to intracellular interacting proteins (Gproteins, β-arrestins, etc.) and will delineate ways to interfere withthese pharmacologically relevant interactions. Efforts to obtain andcrystallize GPCRs are, therefore, of great importance. However, this isa particularly difficult endeavor due to the biochemical challenges inworking with GPCRs and the inherent instability of these complexes indetergent solutions. Also, the intrinsic conformational flexibility ofGPCRs complicates high-resolution structure analysis of GPCRs alonebecause growing diffraction-quality crystals require stable,conformationally homogenous proteins (Kobilka et al. 2007). Provided arenew experimental and analytical tools to capture or “freeze” functionalconformational states of a GPCR of interest, in particular, its activeconformational state, allowing the structural and functional analysis ofthe GPCR, including high resolution structural analysis and manyapplications derived thereof.

Described herein is a protein binding domain that is capable ofspecifically binding to a functional conformational state of a GPCR.

The protein binding domain hereof can be any non-naturally occurringmolecule or part thereof (as defined hereinbefore) that is capable ofspecifically binding to a functional conformational state of a targetGPCR. In one embodiment, the protein binding domains as described hereinare protein scaffolds. The term “protein scaffold” refers generally tofolding units that form structures, particularly protein or peptidestructures, that comprise frameworks for the binding of anothermolecule, for instance, a protein (see, e.g., J. Skerra, 2000, forreview). A protein binding domain can be derived from a naturallyoccurring molecule, e.g., from components of the innate or adaptiveimmune system, or it can be entirely artificially designed. A proteinbinding domain can be immunoglobulin-based or it can be based on domainspresent in proteins including, but limited to, microbial proteins,protease inhibitors, toxins, fibronectin, lipocalins, single chainantiparallel coiled coil proteins or repeat motif proteins. Examples ofprotein binding domains that are known in the art include, but are notlimited to: antibodies, heavy chain antibodies (hcAb), single domainantibodies (sdAb), minibodies, the variable domain derived from camelidheavy chain antibodies (VHH or nanobodies), the variable domain of thenew antigen receptors derived from shark antibodies (VNAR), alphabodies,protein A, protein G, designed ankyrin-repeat domains (DARPins),fibronectin type III repeats, anticalins, knottins, engineered CH2domains (nanoantibodies), peptides and proteins, lipopeptides (e.g.,pepducins), DNA, and RNA (see, e.g., Gebauer & Skerra, 2009; Skerra,2000; Starovasnik et al. 1997; Binz et al. 2004; Koide et al. 1998;Dimitrov, 2009; Nygren et al. 2008; WO2010066740). Frequently, whengenerating a particular type of protein binding domain using selectionmethods, combinatorial libraries comprising a consensus or frameworksequence containing randomized potential interaction residues are usedto screen for binding to a molecule of interest, such as a protein.

“G-protein-coupled receptors,” or “GPCRs,” as used herein, arepolypeptides that share a common structural motif having seven regionsof between 22 to 24 hydrophobic amino acids that form seven alphahelices, each of which spans the membrane. Each span is identified bynumber, i.e., transmembrane-1 (TM1), transmembrane-2 (TM2), etc. Thetransmembrane helices are joined by regions of amino acids betweentransmembrane-2 and transmembrane-3, transmembrane-4 andtransmembrane-5, and transmembrane-6 and transmembrane-7 on theexterior, or “extracellular” side, of the cell membrane, referred to as“extracellular” regions 1, 2 and 3 (EC1, EC2 and EC3), respectively. Thetransmembrane helices are also joined by regions of amino acids betweentransmembrane-1 and transmembrane-2, transmembrane-3 andtransmembrane-4, and transmembrane-5 and transmembrane-6 on theinterior, or “intracellular” side, of the cell membrane, referred to as“intracellular” regions 1, 2 and 3 (IC1, IC2 and IC3), respectively. The“carboxy” (“C”) terminus of the receptor lies in the intracellular spacewithin the cell, and the “amino” (“N”) terminus of the receptor lies inthe extracellular space outside of the cell. Any of these regions arereadily identifiable by analysis of the primary amino acid sequence of aGPCR.

GPCR structure and classification is generally well known in the art andfurther discussion of GPCRs may be found in Probst et al. 1992, Marcheseet al. 1994, Lagerström & Schiöth 2008, Rosenbaum et al. 2009, and thefollowing books: Jurgen Wess (Ed), Structure Function Analysis of GProtein-Coupled Receptors published by Wiley-Liss (first edition; Oct.15, 1999); Kevin R. Lynch (Ed), Identification and Expression of GProtein-Coupled Receptors published by John Wiley & Sons (March 1998);and Tatsuya Haga (Ed), G Protein-Coupled Receptors, published by CRCPress (Sep. 24, 1999); and Steve Watson (Ed) G-Protein Linked ReceptorFactsbook, published by Academic Press (1st edition; 1994).

GPCRs can be grouped on the basis of sequence homology into severaldistinct families. Although all GPCRs have a similar architecture ofseven membrane-spanning α-helices, the different families within thisreceptor class show no sequence homology to one another, thus suggestingthat the similarity of their transmembrane domain structure might definecommon functional requirements. A comprehensive view of the GPCRrepertoire was possible when the first draft of the human genome becameavailable. Fredriksson and colleagues divided 802 human GPCRs intofamilies on the basis of phylogenetic criteria. This showed that most ofthe human GPCRs can be found in five main families, termed Glutamate,Rhodopsin, Adhesion, Frizzled/Taste2 and Secretin (Fredriksson et al.2003).

In a preferred embodiment hereof, the protein binding domain is directedagainst or is capable of specifically binding to a functionalconformational state of a GPCR, wherein the GPCR is chosen from thegroup comprising a GPCR of the Glutamate family of GPCRs, a GPCR of theRhodopsin family of GPCRs, a GPCR of the Adhesion family of GPCRs, aGPCR of the Frizzled/Taste2 family of GPCRs, and a GPCR of the Secretinfamily of GPCRs. Preferably, the GPCR is a mammalian protein, or a plantprotein, or a microbial protein, or a viral protein, or an insectprotein. Even more preferably, the GPCR is a human protein.

Members of the Rhodopsin family (corresponding to class A (Kolakowski,1994) or Class 1 (Foord et al. (2005) in older classification systems)only have small extracellular loops and the interaction of the ligandsoccurs with residues within the transmembrane cleft. This is by far thelargest group (>90% of the GPCRs) and contains receptors for odorants,small molecules such as catecholamines and amines, (neuro)peptides andglycoprotein hormones. Rhodopsin, a representative of this family, isthe first GPCR for which the structure has been solved (Palczewski etal. 2000). β₂AR, the first receptor interacting with a diffusible ligandfor which the structure has been solved (Rosenbaum et al. 2007) alsobelongs to this family. Based on Phylogenetic analysis, class B GPCRs orClass 2 (Foord et al. 2005) receptors have recently been subdivided intotwo families: adhesion and secretin (Fredriksson et al. 2003). Adhesionand secretin receptors are characterized by a relatively long aminoterminal extracellular domain involved in ligand-binding. Little isknown about the orientation of the transmembrane domains, but it isprobably quite different from that of rhodopsin. Ligands for these GPCRsare hormones, such as glucagon, secretin, gonadotropin-releasing hormoneand parathyroid hormone. The Glutamate family receptors (Class C orClass 3 receptors) also have a large extracellular domain, whichfunctions like a “Venus fly trap” since it can open and close with theagonist bound inside. Family members are the metabotropic glutamate, theCa²⁺-sensing and the γ-aminobutyric acid (GABA)-B receptors.

GPCRs include, without limitation, serotonin olfactory receptors,glycoprotein hormone receptors, chemokine receptors, adenosinereceptors, biogenic amine receptors, melanocortin receptors,neuropeptide receptors, chemotactic receptors, somatostatin receptors,opioid receptors, melatonin receptors, calcitonin receptors, PTH/PTHrPreceptors, glucagon receptors, secretin receptors, latrotoxin receptors,metabotropic glutamate receptors, calcium receptors, GABA-B receptors,pheromone receptors, the protease-activated receptors, the rhodopsinsand other G-protein-coupled seven transmembrane segment receptors. GPCRsalso include these GPCR receptors associated with each other ashomomeric or heteromeric dimers or as higher-order oligomers. The aminoacid sequences (and the nucleotide sequences of the cDNAs that encodethem) of GPCRs are readily available, for example by reference toGenBank (on the World Wide Web at ncbi.nlm.nih.gov/entrez).

According to a preferred embodiment, the GPCR is chosen from the groupcomprising the adrenergic receptors, preferably the α-adrenergicreceptors, such as the α₁-adrenergic receptors and the α₂-adrenergicreceptors, and the β-adrenergic receptors, such as the β₁-adrenergicreceptors, the β₂-adrenergic receptors and the β₃-adrenergic receptors;or from the group comprising the muscarinic receptors, preferably theM₁-muscarinic receptors, the M₂-muscarinic receptors, the M₃-muscarinicreceptors, the M₄-muscarinic receptors and the M₅-muscarinic receptors;or from the group of the angiotensin receptors, preferably theangiotensin II type 1 receptor, the angiotensin II type 2 receptor andother atypical angiotensin II receptors; all of which are well known inthe art.

A GPCR, as used herein, may be any naturally occurring or non-naturallyoccurring (i.e., altered by man) polypeptide. The term “naturallyoccurring” in reference to a GPCR means a GPCR that is naturallyproduced (for example and without limitation, by a mammal, morespecifically by a human, or by a virus, or by a plant, or by an insect,amongst others). Such GPCRs are found in nature. The term “non-naturallyoccurring” in reference to a GPCR means a GPCR that is not naturallyoccurring. Wild-type GPCRs that have been made constitutively activethrough mutation, and variants of naturally occurring GPCRs are examplesof non-naturally occurring GPCRs. Non-naturally occurring GPCR may havean amino acid sequence that is at least 80% identical to, at least 90%identical to, at least 95% identical to or at least 99% identical to, anaturally occurring GPCR. Taking the β₂-adrenergic receptor as aparticular non-limiting example of a GPCR within the scope hereof, itshould be clear from the above that in addition to the humanβ₂-adrenergic receptor (e.g., the sequence described by Genbankaccession number NP_000015), the mouse β₂-adrenergic receptor (e.g., asdescribed by Genbank accession no. NM 007420) or other mammalianβ₂-adrenergic receptor may also be employed. In addition, the term isintended to encompass wild-type polymorphic variants and certain otheractive variants of the β₂-adrenergic receptor from a particular species.For example, a “human β₂-adrenergic receptor” has an amino acid sequencethat is at least 95% identical to (e.g., at least 95% or at least 98%identical to) the naturally occurring “human β₂-adrenoreceptor” ofGenbank accession number NP 000015.

Further, it will be appreciated that the disclosure also envisages GPCRswith a loop deletion, or an N- and/or C-terminal deletion, or asubstitution, or an insertion or addition in relation to its amino acidor nucleotide sequence, or any combination thereof (as definedhereinbefore, and see also Example section). It is further expected thatthe protein binding domains hereof will generally be capable of bindingto all naturally occurring or synthetic analogs, variants, mutants, oralleles of the GPCR.

Various methods may be used to determine specific binding between theprotein binding domain and a target GPCR, including, for example, enzymelinked immunosorbent assays (ELISA), surface Plasmon resonance assays,phage display, and the like, which are common practice in the art, forexample, and discussed in Sambrook et al. (2001), Molecular Cloning, ALaboratory Manual, Third Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. It will be appreciated that for this purpose, aunique label or tag will often be used, such as a peptide label, anucleic acid label, a chemical label, a fluorescent label, or a radiofrequency tag, as described further herein.

It should be clear that GPCRs are conformationally complex membraneproteins that exhibit a spectrum-functional behavior in response tonatural and synthetic ligands. Defining the pathway from agonist bindingto protein activation will require a combination of crystal structuresof different conformational states of the receptor under investigationin complex with various natural or synthetic ligands (includingstructures of active agonist-bound states and GPCR-G protein complexes),which will provide snapshots along the activation pathway.

Thus, in a preferred embodiment, the protein binding domain is capableof stabilizing or otherwise increasing the stability of a particularfunctional conformational state of a GPCR. Preferably, the proteinbinding domain is capable of inducing the formation of a functionalconformational state in a GPCR upon binding the GPCR. The functionalconformation state of the GPCR can be a basal conformational state, oran active conformational state or an inactive conformational state.Preferably, the protein binding domain is capable of stabilizing a GPCRin its active conformational state and/or is capable of forcing the GPCRto adopt its active conformational state upon binding.

The wording “inducing” or “forcing” or “locking” or “trapping” or“fixing” or “freezing” with respect to a functional conformational stateof a GPCR (as defined herein), as used herein, refers to the retainingor holding of a GPCR in a subset of the possible conformations that itcould otherwise assume, due to the effects of the interaction of theGPCR with the protein binding domain hereof. Accordingly, a protein thatis “conformationally trapped” or “conformationally fixed” or“conformationally locked” or “conformationally frozen,” as used herein,is one that is held in a subset of the possible conformations that itcould otherwise assume, due to the effects of the interaction of theGPCR with the protein binding domain hereof. Within this context, aprotein binding domain that specifically or selectively binds to aspecific conformation or conformational state of a protein refers to aprotein binding domain that binds with a higher affinity to a protein ina subset of conformations or conformational states than to otherconformations or conformational states that the protein may assume. Oneof skill in the art will recognize that protein binding domains thatspecifically or selectively bind to a specific conformation orconformational state of a protein will stabilize this specificconformation or conformational state.

The term “a functional conformational state,” as used herein, refers tothe fact that proteins, in particular, membrane proteins such as GPCRs,possess many different conformational states having a dynamic range ofactivity, in particular, ranging from no activity to maximal activity(reviewed in Kobilka and Deupi, 2007). It should be clear that “afunctional conformational state” is not meant to cover the denaturedstates of proteins. The functional versatility of GPCRs is inherentlycoupled to the flexibility of these proteins resulting in such aspectrum of conformations. The conformational energy landscape isintrinsically coupled to such factors as the presence of bound ligands(effector molecules, agonists, antagonists, inverse agonists, etc.), thelipid environment or the binding of interacting proteins. For example, a“basal conformational state” can be defined as a low energy state of thereceptor in the absence of a ligand (as defined hereinbefore, e.g.,effector molecules, agonists, antagonists, inverse agonists).

The probability that a protein will undergo a transition to anotherconformational state is a function of the energy difference between thetwo states and the height of the energy barrier between the two states.In the case of a receptor protein, such as a GPCR, the energy of ligandbinding can be used either to alter the energy barrier between the twostates, or to change the relative energy levels between the two states,or both. Changing of the energy barrier would have an effect on the rateof transition between the two states, whereas changing the energy levelswould have an effect on the equilibrium distribution of receptors in twostates. Binding of an agonist or partial agonist would lower the energybarrier and/or reduce the energy of the more active conformational staterelative to the inactive conformational state. An inverse agonist wouldincrease the energy barrier and/or reduce the energy of the “inactivestate conformation” relative to the “active conformation.” Coupling ofthe receptor to its G protein could further alter the energy landscape.The activities of integral membrane proteins, including GPCRs, are alsoaffected by the structures of the lipid molecules that surround them inthe membrane. Membrane proteins are not rigid entities, and deform toensure good hydrophobic matching to the surrounding lipid bilayer. Oneimportant parameter is the hydrophobic thickness of the lipid bilayer,defined by the lengths of the lipid fatty acyl chains. Also, thestructure of the lipid headgroup region is likely to be important indefining the structures of those parts of a membrane protein that arelocated in the lipid headgroup region. Among other lipids,palmitoylation and binding of cholesterol to GPCRs may also play astructural role inside monomeric receptors and contribute to theformation/stabilization of receptor oligomers (Lee 2004; Chini andParenti 2009).

“Receptor ligands,” or simply “ligands,” as defined hereinbefore, may be“orthosteric” ligands (both natural and synthetic), meaning that theybind to the receptor's active site and are further classified accordingto their efficacy or, in other words, to the effect they have onreceptor signaling through a specific pathway. As used herein, an“agonist” refers to a ligand that, by binding a receptor, increases thereceptor's signaling activity. Full agonists are capable of maximalreceptor stimulation; partial agonists are unable to elicit fullactivity even at saturating concentrations. Partial agonists can alsofunction as “blockers” by preventing the binding of more robustagonists. An “antagonist” refers to a ligand that binds a receptorwithout stimulating any activity. An “antagonist” is also known as a“blocker” because of its ability to prevent binding of other ligandsand, therefore, block agonist-induced activity. Further, an “inverseagonist” refers to an antagonist that, in addition to blocking agonisteffects, reduces receptors' basal or constitutive activity below that ofthe unliganded receptor.

The canonical view of how GPCRs regulate cellular physiology is that thebinding of ligands (such as hormones, neurotransmitters or sensorystimuli) stabilizes an active conformational state of the receptor,thereby allowing interactions with heterotrimeric G proteins. Inaddition to interacting with G proteins, agonist-bound GPCRs associatewith GPCR kinases (GRKs), leading to receptor phosphorylation. A commonoutcome of GPCR phosphorylation by GRKs is a decrease in GPCRinteractions with G proteins and an increase in GPCR interactions witharrestins, which sterically interdict further G-protein signaling,resulting in receptor desensitization. As β-arrestins turn off G-proteinsignals, they can simultaneously initiate a second, parallel set ofsignal cascades, such as the MAPK pathway. GPCRs also associate withvarious proteins outside the families of general GPCR-interactingproteins (G proteins, GRKs, arrestins and other receptors). TheseGPCR-selective partners can mediate GPCR signaling, organize GPCRsignaling through G proteins, direct GPCR trafficking, anchor GPCRs, inparticular, subcellular areas and/or influence GPCR pharmacology (Ritterand Hall 2009). In this regard, “ligands,” as used herein, may also be“biased ligands” with the ability to selectively stimulate a subset of areceptor's signaling activities, for example, the selective activationof G-protein or β-arrestin function. Such ligands are known as “biasedligands,” “biased agonists” or “functionally selective agonists.” Moreparticularly, ligand bias can be an imperfect bias characterized by aligand stimulation of multiple receptor activities with differentrelative efficacies for different signals (non-absolute selectivity) orcan be a perfect bias characterized by a ligand stimulation of onereceptor activity without any stimulation of another known receptoractivity.

The signaling activities of GPCRs (and thus their conformationalbehavior) may also be affected by the binding of another kind of ligandsknown as allosteric regulators. “Allosteric regulators” or otherwise“allosteric modulators,” “allosteric ligands” or “effector molecules”bind at an allosteric site of a GPCR (that is, a regulatory sitephysically distinct from the protein's active site). In contrast toorthosteric ligands, allosteric modulators are non-competitive becausethey bind receptors at a different site and modify receptor functioneven if the endogenous ligand also is binding. Because of this,allosteric modulators are not limited to simply turning a receptor on oroff, the way most drugs are. Instead, they act more like a dimmerswitch, offering control over the intensity of activation ordeactivation, while allowing the body to retain its natural control overinitiating receptor activation. Allosteric regulators that enhance theprotein's activity are referred to herein as “allosteric activators” or“positive allosteric modulators,” whereas, those that decrease theprotein's activity are referred to herein as “allosteric inhibitors” orotherwise “negative allosteric modulators.”

Preferably, the protein binding domain hereof is capable of specificallybinding to an agonist-bound GPCR and/or enhances the affinity of a GPCRfor an agonist. It is preferred that the protein binding domain iscapable of increasing the affinity for the agonist at least two-fold, atleast five-fold and, more preferably, at least ten-fold upon binding tothe receptor as measured by a decrease in EC₅₀, IC₅₀, K_(d), or anyother measure of affinity or potency known to one of skill in the art.

It will be appreciated that having increased stability with respect tostructure and/or a particular biological activity of a GPCR may also bea guide to the stability of other denaturants or denaturing conditionsincluding heat, a detergent, a chaotropic agent and an extreme pH.Accordingly, in a further embodiment, the protein binding domain hereofis capable of increasing the stability of a functional conformationalstate of a GPCR under non-physiological conditions induced by dilution,concentration, buffer composition, heating, cooling, freezing,detergent, chaotropic agent, or pH. In contrast to water-solubleproteins, thermodynamic studies of membrane protein folding andstability have proven to be extremely challenging, and complicated bythe difficulty of finding conditions for reversible folding. Unfoldingof helical membrane proteins induced by most methods, such as thermaland chemical approaches, is irreversible as reviewed by Stanley andFleming (2008). The term “thermostabilize,” “thermostabilizing,”“increasing the thermostability of,” as used herein, therefore, refersto the functional rather than to the thermodynamic properties of a GPCRand to the protein's resistance to irreversible denaturation induced bythermal and/or chemical approaches including, but not limited to,heating, cooling, freezing, chemical denaturants, pH, detergents, salts,additives, proteases or temperature. Irreversible denaturation leads tothe irreversible unfolding of the functional conformations of theprotein, loss of biological activity and aggregation of the denaturatedprotein. The terms “(thermo)stabilize,” “(thermo)stabilizing,”“increasing the (thermo)stability of,” as used herein, apply to GPCRsembedded in lipid particles or lipid layers (for example, lipidmonolayers, lipid bilayers, and the like) and to GPCRs that have beensolubilized in detergent.

Preferably, the protein binding domain hereof is capable of increasingthe thermostability of a functional conformational state of a GPCR,preferably, an active conformational state of a GPCR. In relation to anincreased stability to heat, this can be readily determined by measuringligand binding or by using spectroscopic methods such as fluorescence,CD or light scattering that are sensitive to unfolding at increasingtemperatures (see also Example section). It is preferred that theprotein binding domain is capable of increasing the stability asmeasured by an increase in the thermal stability of a GPCR in afunctional conformational state with at least 2° C., at least 5° C., atleast 8° C., and more preferably at least 10° C. or 15° C. or 20° C.According to another preferred embodiment, the protein binding domain iscapable of increasing the thermal stability of a functional conformationof a GPCR in complex with a ligand such as, but not restricted to, anagonist, an inverse agonist, an antagonist and/or a modulator or aninhibitor of the GPCR or the GPCR-dependent signaling pathway. Accordingto another preferred embodiment, the protein binding domain hereof iscapable of increasing the stability in the presence of a detergent or achaotrope of a functional conformational state of a GPCR. Preferably,the protein binding domain is capable of increasing the stability todenaturation induced by thermal or chemical approaches of the activeconformational state of a GPCR. In relation to an increased stability toheat a detergent or to a chaotrope, the GPCR is typically incubated fora defined time in the presence of a test detergent or a test chaotropicagent and the stability is determined using, for example, ligand bindingor a spectroscoptic method, optionally at increasing temperatures asdiscussed above. According to still another preferred embodiment, theprotein binding domain hereof is capable of increasing the stability toextreme pH of a functional conformational state of a GPCR. Preferably,the protein binding domain is capable of increasing the stability toextreme pH of the active conformational state of a GPCR. In relation toan extreme of pH, a typical test pH would be chosen, for example, in therange 6 to 8, the range 5.5 to 8.5, the range 5 to 9, the range 4.5 to9.5, more specifically in the range 4.5 to 5.5 (low pH) or in the range8.5 to 9.5 (high pH).

The protein binding domains hereof may generally be directed against anydesired GPCR and may, in particular, be directed against anyconformational epitope of any GPCR, preferably a functionalconformational state of any GPCR, more preferably an activeconformational state of a GPCR (all as defined hereinbefore). Moreparticularly, the conformational epitope can be part of an intracellularor extracellular region, or an intramembranous region, or a domain orloop structure of any desired GPCR. According to particular embodiments,the protein binding domains may be directed against any suitableextracellular region, domain, loop or other extracellular conformationalepitope of a GPCR, but is preferably directed against one of theextracellular parts of the transmembrane domains or more preferablyagainst one of the extracellular loops that link the transmembranedomains. Alternatively, the protein binding domains may be directedagainst any suitable intracellular region, domain, loop or otherintracellular conformational epitope of a GPCR, but is preferablydirected against one of the intracellular parts of the transmembranedomains or, more preferably, against one of the intracellular loops thatlink the transmembrane domains. A protein binding domain thatspecifically binds to a “three-dimensional” epitope or “conformational”epitope specifically binds to a tertiary (i.e., three-dimensional)structure of a folded protein, and binds at much reduced (i.e., by afactor of at least 2, 5, 10, 50 or 100) affinity to the linear (i.e.,unfolded, denatured) form of the protein. It is further expected thatthe protein binding domains hereof will generally bind to all naturallyoccurring or synthetic analogs, variants, mutants, or alleles of theGPCR.

In a specific embodiment, the protein binding domain hereof is capableof specifically binding to an intracellular conformational epitope of aGPCR. Preferably, the protein binding domain is capable of specificallybinding a conformational epitope that is comprised in, located at, oroverlaps with, the G protein binding site of a GPCR. More preferably,the protein binding domains may occupy the G protein binding site of afunctional conformational state of a GPCR, more preferably, of an activeconformational state of a GPCR. Most preferably, the protein bindingdomains show G protein—like behavior. The term “G protein-like behavior”as used herein refers to the property of protein binding domains topreferentially bind agonist-bound receptor versus, for example, inverseagonist-bound receptor. Protein binding domains showing G protein-likebehavior also enhance the affinity of the receptor for agonists, whichis attributed to the cooperative interaction between agonist-occupiedreceptor and G protein (see also Example section).

In a preferred embodiment, the protein binding domain is derived from aninnate or adaptive immune system. Preferably, the protein binding domainis derived from an immunoglobulin. Preferably, the protein bindingdomain hereof is an antibody or a derivative thereof. The term“antibody” (Ab) refers generally to a polypeptide encoded by animmunoglobulin gene, or functional fragments thereof, that specificallybinds and recognizes an antigen, and is known to the person skilled inthe art. A conventional immunoglobulin (antibody) structural unitcomprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one “light” (about 25 kDa) andone “heavy” chain (about 50-70 kDa). The N-terminus of each chaindefines a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The terms variable lightchain (VL) and variable heavy chain (VH) refer to these light and heavychains, respectively. The term “antibody” is meant to include wholeantibodies, including single-chain whole antibodies, and antigen-bindingfragments. In some embodiments, antigen-binding fragments may beantigen-binding antibody fragments that include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (dsFv) and fragments comprising orconsisting of either a VL or VH domain, and any combination of those orany other functional portion of an immunoglobulin peptide capable ofbinding to the target antigen. The term “antibodies” is also meant toinclude heavy chain antibodies, or functional fragments thereof, such assingle domain antibodies, more specifically, nanobodies, as definedfurther herein.

Preferably, the protein binding domain comprises an amino acid sequencecomprising four framework regions and three complementarity-determiningregions, preferably in a sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, or anysuitable fragment thereof (which will then usually contain at least someof the amino acid residues that form at least one of thecomplementarity-determining regions). Protein binding domains comprisingfour FRs and three CDRs are known to the person skilled in the art andhave been described, as a non-limiting example, in Wesolowski et al.(2009).

Preferably, the protein binding domain hereof is derived from a camelidantibody. More preferably, the protein binding domain hereof comprisesan amino acid sequence of a nanobody, or any suitable fragment thereof.More specifically, the protein binding domain is a nanobody or anysuitable fragment thereof. A “nanobody” (Nb), as used herein, refers tothe smallest antigen binding fragment or single variable domain (“VHH”)derived from naturally occurring heavy chain antibody and is known tothe person skilled in the art. They are derived from heavy chain onlyantibodies, seen in camelids (Hamers-Casterman et al. 1993; Desmyter etal. 1996). In the family of “camelids,” immunoglobulins devoid of lightpolypeptide chains are found. “Camelids” comprise old world camelids(Camelus bactrianus and Camelus dromedarius) and new world camelids (forexample, Lama paccos, Lama glama, Lama guanicoe and Lama vicugna). Thesingle variable domain heavy chain antibody is herein designated as ananobody or a VHH antibody. Nanobody™, Nanobodies™ and Nanoclone™ aretrademarks of Ablynx NV (Belgium). The small size and unique biophysicalproperties of Nbs excel conventional antibody fragments for therecognition of uncommon or hidden epitopes and for binding into cavitiesor active sites of protein targets. Further, Nbs can be designed asbispecific and bivalent antibodies or attached to reporter molecules(Conrath et al. 2001). Nbs are stable and rigid single domain proteinsthat can easily be manufactured and survive the gastro-intestinalsystem. Therefore, Nbs can be used in many applications including drugdiscovery and therapy (Saerens et al. 2008), but also as a versatile andvaluable tool for purification, functional study and crystallization ofproteins (Conrath et al. 2009).

The nanobodies hereof generally comprise a single amino acid chain thatcan be considered to comprise four “framework sequences” or FRs andthree “complementarity-determining regions” or CDRs (as definedhereinbefore). Non-limiting examples of nanobodies hereof are describedin more detail further herein. It should be clear that framework regionsof nanobodies may also contribute to the binding of their antigens(Desmyter et al. 2002; Korotkov et al. 2009).

Non-limiting examples of the nanobodies hereof include, but are notlimited to, nanobodies as defined by SEQ ID NOS:1-29 (see FIG. 12, Table1). The delineation of the CDR sequences is based on the IMGT uniquenumbering system for V-domains and V-like domains (Lefranc et al. 2003).In a specific embodiment, the above nanobodies can comprise at least oneof the complementarity-determining regions (CDRs) with an amino acidsequence selected from SEQ ID NOS:30-70 (see FIG. 12; Table 2). Morespecifically, the above nanobodies can be selected from the groupcomprising SEQ ID NOS:1-29, or a functional fragment thereof. A“functional fragment” or a “suitable fragment,” as used herein, may, forexample, comprise one of the CDR loops. Preferably, the functionalfragment comprises CDR3. More specifically, the nanobodies consist ofany of SEQ ID NOS:1-29 and the functional fragment of the nanobodiesconsist of any of SEQ ID NOS:30-70. In still another embodiment, anucleic acid sequence encoding any of the above nanobodies or functionalfragments is also part hereof. Further, also envisaged are expressionvectors comprising nucleic acid sequences encoding any of the abovenanobodies or functional fragments thereof, as well as host cellsexpressing such expression vectors. Suitable expression systems includeconstitutive and inducible expression systems in bacteria or yeasts,virus expression systems, such as baculovirus, semliki forest virus andlentiviruses, or transient transfection in insect or mammalian cells.Suitable host cells include E. coli, Lactococcus lactis, Saccharomycescerevisiae, Schizosaccharomyces pombe, Pichia pastoris, and the like.Suitable animal host cells include HEK 293, COS, S2, CHO, NSO, DT40 andthe like. The cloning, expression and/or purification of the nanobodiescan be done according to techniques known by the skilled person in theart.

It should be noted that the term “nanobody,” as used herein in itsbroadest sense, is not limited to a specific biological source or to aspecific method of preparation. For example, the nanobodies hereof cangenerally be obtained: (1) by isolating the VHH domain of a naturallyoccurring heavy chain antibody; (2) by expression of a nucleotidesequence encoding a naturally occurring VHH domain; (3) by“humanization” of a naturally occurring VHH domain or by expression of anucleic acid encoding such a humanized VHH domain; (4) by “camelization”of a naturally occurring VH domain from any animal species, and, inparticular, from a mammalian species, such as from a human being, or byexpression of a nucleic acid encoding such a camelized VH domain; (5) by“camelization” of a “domain antibody” or “Dab” as described in the art,or by expression of a nucleic acid encoding such a camelized VH domain;(6) by using synthetic or semi-synthetic techniques for preparingproteins, polypeptides or other amino acid sequences known per se; (7)by preparing a nucleic acid encoding a nanobody using techniques fornucleic acid synthesis known per se, followed by expression of thenucleic acid thus obtained; and/or (8) by any combination of one or moreof the foregoing.

One preferred class of nanobodies corresponds to the VHH domains ofnaturally occurring heavy chain antibodies directed against a functionalconformational state of a GPCR. Although naive or synthetic libraries ofnanobodies (for examples of such libraries, see WO9937681, WO0043507,WO0190190, WO03025020 and WO03035694) may contain conformational bindersagainst a GPCR in a functional conformational state, a preferredembodiment of this invention includes the immunization of a Camelidaewith a GPCR in a functional conformational state, optionally bound to areceptor ligand, to expose the immune system of the animal with theconformational epitopes that are unique to the GPCR (for example,agonist-bound GPCR so as to raise antibodies directed against a GPCR inits active conformational state). Thus, as further described herein,such VHH sequences can preferably be generated or obtained by suitablyimmunizing a species of Camelid with a GPCR, preferably a GPCR in afunctional conformational state, more preferably an activeconformational state (i.e., so as to raise an immune response and/orheavy chain antibodies directed against the GPCR), by obtaining asuitable biological sample from the Camelid (such as a blood sample, orany sample of B-cells), and by generating VHH sequences directed againstthe GPCR, starting from the sample. Such techniques will be clear to theskilled person. Yet another technique for obtaining the desired VHHsequences involves suitably immunizing a transgenic mammal that iscapable of expressing heavy chain antibodies (i.e., so as to raise animmune response and/or heavy chain antibodies directed against a GPCR ina functional conformational state), obtaining a suitable biologicalsample from the transgenic mammal (such as a blood sample, or any sampleof B-cells), and then generating VHH sequences directed against the GPCRstarting from the sample, using any suitable technique known per se. Forexample, for this purpose, the heavy chain antibody-expressing mice andthe further methods and techniques described in WO02085945 and inWO04049794 can be used.

Accordingly, the invention encompasses methods of generating proteinbinding domains hereof. As a non-limiting example, a method is providedof generating nanobodies specifically binding to a conformationalepitope of a functional conformational state of a GPCR, comprising:

-   -   (i) immunizing an animal with a GPCR, and    -   (ii) screening for nanobodies specifically binding to a        conformational epitope of a functional conformational state of        the GPCR.

Preferably, immunization of an animal will be done with a GPCR in thepresence of a receptor ligand, wherein the ligand induces a particularfunctional conformational state of the GPCR. For example, nanobodies maybe generated that are specifically binding to a conformational epitopeof an active conformational state of a GPCR by immunizing an animal witha GPCR in the presence of an agonist that induces the formation of anactive conformational state of the GPCR (see also Example section).

For the immunization of an animal with a GPCR, the GPCR may be producedand purified using conventional methods that may employ expressing arecombinant form of the GPCR in a host cell, and purifying the GPCRusing affinity chromatography and/or antibody-based methods. Inparticular embodiments, the baculovirus/Sf-9 system may be employed forexpression, although other expression systems (e.g., bacterial, yeast ormammalian cell systems) may also be used. Exemplary methods forexpressing and purifying GCPRs are described in, for example, Kobilka(1995), Eroglu et al. (2002), Chelikani et al. (2006) and the bookIdentification and Expression of G Protein-Coupled Receptors (Kevin R.Lynch (Ed.), 1998), among many others. A GPCR may also be reconstitutedin phospholipid vesicles. Likewise, methods for reconstituting an activeGPCR in phospholipid vesicles are known, and are described in: Luca etal. (2003), Mansoor et al. (2006), Niu et al. (2005), Shimada et al.(2002), and Eroglu et al. (2003), among others. In certain cases, theGPCR and phospholipids may be reconstituted at high density (e.g., 1 mgreceptor per mg of phospholipid). In particular embodiments, thephospholipids vesicles may be tested to confirm that the GPCR is active.In many cases, a GPCR may be present in the phospholipid vesicle in bothorientations (in the normal orientation, and in the “upside down”orientation in which the intracellular loops are on the outside of thevesicle). Other immunization methods with a GPCR include, withoutlimitation, the use of complete cells expressing a GPCR, vaccinationwith a nucleic acid sequence encoding a GPCR (e.g., DNA vaccination),immunization with viruses or virus-like particles expressing a GPCR,amongst others.

Any suitable animal, e.g., a warm-blooded animal, in particular, amammal such as a rabbit, mouse, rat, camel, sheep, cow, shark, or pig ora bird such as a chicken or turkey, may be immunized using any of thetechniques well known in the art suitable for generating an immuneresponse.

The screening for nanobodies, as a non-limiting example, specificallybinding to a conformational epitope of a functional conformational stateof the GPCR may, for example, be performed by screening a set,collection or library of cells that express heavy chain antibodies ontheir surface (e.g., B-cells obtained from a suitably immunizedCamelid), or bacteriophages that display a fusion of genIII and nanobodyat their surface, by screening of a (naïve or immune) library of VHHsequences or nanobody sequences, or by screening of a (naïve or immune)library of nucleic acid sequences that encode VHH sequences or nanobodysequences, which may all be performed in a manner known per se, andwhich method may optionally further comprise one or more other suitablesteps, such as, for example and without limitation, a step of affinitymaturation, a step of expressing the desired amino acid sequence, a stepof screening for binding and/or for activity against the desired antigen(in this case, the GPCR), a step of determining the desired amino acidsequence or nucleotide sequence, a step of introducing one or morehumanizing substitutions, a step of formatting in a suitable multivalentand/or multispecific format, a step of screening for the desiredbiological and/or physiological properties (i.e., using a suitable assayknown in the art), and/or any combination of one or more of such steps,in any suitable order.

A particularly preferred class of protein binding domains hereofcomprises nanobodies with an amino acid sequence that corresponds to theamino acid sequence of a naturally occurring VHH domain, but that hasbeen “humanized,” i.e., by replacing one or more amino acid residues inthe amino acid sequence of the naturally occurring VHH sequence (and, inparticular, in the framework sequences) by one or more of the amino acidresidues that occur at the corresponding position(s) in a VH domain froma conventional four-chain antibody from a human being. This can beperformed in a manner known per se, which will be clear to the skilledperson, on the basis of the further description herein and the prior arton humanization. Again, it should be noted that such humanizedNanobodies hereof can be obtained in any suitable manner known per se(i.e., as indicated under points (1)-(8) above) and, thus, are notstrictly limited to polypeptides that have been obtained using apolypeptide that comprises a naturally occurring VHH domain as astarting material. Humanized nanobodies may have several advantages,such as a reduced immunogenicity, compared to the correspondingnaturally occurring VHH domains. Such humanization generally involvesreplacing one or more amino acid residues in the sequence of a naturallyoccurring VHH with the amino acid residues that occur at the sameposition in a human VH domain, such as a human VH3 domain. Thehumanizing substitutions should be chosen such that the resultinghumanized nanobodies still retain the favorable properties of nanobodiesas defined herein. The skilled person will be able to select humanizingsubstitutions or suitable combinations of humanizing substitutions thatoptimize or achieve a desired or suitable balance between the favorableproperties provided by the humanizing substitutions on the one hand andthe favorable properties of naturally occurring VHH domains on the otherhand.

Another particularly preferred class of protein binding domains hereofcomprises nanobodies with an amino acid sequence that corresponds to theamino acid sequence of a naturally occurring VH domain, but that hasbeen “camelized,” i.e., by replacing one or more amino acid residues inthe amino acid sequence of a naturally occurring VH domain from aconventional four-chain antibody by one or more of the amino acidresidues that occur at the corresponding position(s) in a VHH domain ofa heavy chain antibody. Such “camelizing” substitutions are preferablyinserted at amino acid positions that form and/or are present at theVH-VL interface, and/or at the so-called Camelidae hallmark residues, asdefined herein (see, for example, WO9404678). Preferably, the VHsequence that is used as a starting material or starting point forgenerating or designing the camelized nanobody is preferably a VHsequence from a mammal, more preferably the VH sequence of a humanbeing, such as a VH3 sequence. However, it should be noted that suchcamelized nanobodies hereof can be obtained in any suitable manner knownper se (i.e., as indicated under points (1)-(8) above) and thus are notstrictly limited to polypeptides that have been obtained using apolypeptide that comprises a naturally occurring VH domain as a startingmaterial.

For example, both “humanization” and “camelization” can be performed byproviding a nucleotide sequence that encodes a naturally occurring VHHdomain or VH domain, respectively, and then changing, in a manner knownper se, one or more codons in the nucleotide sequence in such a way thatthe new nucleotide sequence encodes a “humanized” or “camelized”nanobody hereof, respectively. This nucleic acid can then be expressedin a manner known per se, so as to provide the desired nanobody hereof.Alternatively, based on the amino acid sequence of a naturally occurringVHH domain or VH domain, respectively, the amino acid sequence of thedesired humanized or camelized nanobody hereof, respectively, can bedesigned and then synthesized de novo using techniques for peptidesynthesis known per se. Also, based on the amino acid sequence ornucleotide sequence of a naturally occurring VHH domain or VH domain,respectively, a nucleotide sequence encoding the desired humanized orcamelized nanobody hereof, respectively, can be designed and thensynthesized de novo using techniques for nucleic acid synthesis knownper se, after which the nucleic acid thus obtained can be expressed in amanner known per se, so as to provide the desired nanobody hereof.

Other suitable methods and techniques for obtaining the nanobodieshereof and/or nucleic acids encoding the same, starting from naturallyoccurring VH sequences or preferably VHH sequences, will be clear fromthe skilled person, and may, for example, comprise combining one or moreparts of one or more naturally occurring VH sequences (such as one ormore FR sequences and/or CDR sequences), one or more parts of one ormore naturally occurring VHH sequences (such as one or more FR sequencesor CDR sequences), and/or one or more synthetic or semi-syntheticsequences, in a suitable manner, so as to provide a nanobody hereof or anucleotide sequence or nucleic acid encoding the same.

It is also within the scope hereof to use natural or synthetic analogs,mutants, variants, alleles, homologs and orthologs (herein collectivelyreferred to as “analogs”) of the protein binding domains hereof,preferably to the nanobodies, and in particular analogs of thenanobodies of SEQ ID NOS:1-29 (see Table 1, FIG. 12). Thus, according toone embodiment hereof, the term “nanobody hereof” in its broadest sensealso covers such analogs. Generally, in such analogs, one or more aminoacid residues may have been replaced, deleted and/or added, compared tothe nanobodies hereof as defined herein. Such substitutions, insertions,deletions or additions may be made in one or more of the frameworkregions and/or in one or more of the CDRs and, in particular, analogs ofthe CDRs of the nanobodies of SEQ ID NOS:1-29, the CDRs correspondingwith SEQ ID NOS:30-70 (see Table 2, FIG. 12). “Analogs,” as used herein,are sequences wherein each or any framework region and each or anycomplementarity-determining region shows at least 80% identity,preferably at least 85% identity, more preferably 90% identity, evenmore preferably, 95% identity with the corresponding region in thereference sequence (i.e., FR1 analog versus FR1 reference, CDR1 analogversus CDR1 reference, FR2 analog versus FR2 reference, CDR2 analogversus CDR2 reference, FR3 analog versus FR3 reference, CDR3 analogversus CDR3 reference, FR4 analog versus FR4 reference), as measured ina BLASTp alignment (Altschul et al. 1987; FR and CDR definitionsaccording to IMGT unique numbering system for V-domains and V-likedomains (Lefranc et al. 2003)).

By means of non-limiting examples, a substitution may, for example, be aconservative substitution (as described herein) and/or an amino acidresidue may be replaced by another amino acid residue that naturallyoccurs at the same position in another VHH domain. Thus, any one or moresubstitutions, deletions or insertions, or any combination thereof, thateither improve the properties of the nanobody hereof or that at least donot detract too much from the desired properties or from the balance orcombination of desired properties of the nanobody hereof (i.e., to theextent that the nanobody is no longer suited for its intended use), areincluded within the scope hereof. A skilled person will generally beable to determine and select suitable substitutions, deletions,insertions, additions, or suitable combinations thereof, based on thedisclosure herein and optionally after a limited degree of routineexperimentation, which may, for example, involve introducing a limitednumber of possible substitutions and determining their influence on theproperties of the nanobodies thus obtained.

For example, and depending on the host organism used to express theprotein binding domain hereof, preferably the nanobody, such deletionsand/or substitutions may be designed in such a way that one or moresites for post-translational modification (such as one or moreglycosylation sites) are removed, as will be within the ability of theperson skilled in the art. Alternatively, substitutions or insertionsmay be designed so as to introduce one or more sites for attachment offunctional groups (as described herein), for example, to allowsite-specific pegylation.

Examples of modifications, as well as examples of amino acid residueswithin the protein binding domain sequence, preferably the nanobodysequence, that can be modified (i.e., either on the protein backbone butpreferably on a side chain), methods and techniques that can be used tointroduce such modifications and the potential uses and advantages ofsuch modifications will be clear to the skilled person. For example,such a modification may involve the introduction (e.g., by covalentlinking or in another suitable manner) of one or more functional groups,residues or moieties into or onto the nanobody hereof invention and, inparticular, of one or more functional groups, residues or moieties thatconfer one or more desired properties or functionalities to the nanobodyhereof. Examples of such functional groups and of techniques forintroducing them will be clear to the skilled person, and can generallycomprise all functional groups and techniques mentioned in the generalbackground art cited hereinabove as well as the functional groups andtechniques known per se for the modification of pharmaceutical proteinsand, in particular, for the modification of antibodies or antibodyfragments (including ScFvs and single domain antibodies), for whichreference is, for example, made to Remington's Pharmaceutical Sciences,16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functionalgroups may, for example, be linked directly (for example, covalently) toa nanobody hereof, or optionally via a suitable linker or spacer, aswill again be clear to the skilled person. One of the most widely usedtechniques for increasing the half-life and/or reducing immunogenicityof pharmaceutical proteins comprises attachment of a suitablepharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG)or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG).Generally, any suitable form of pegylation can be used, such as thepegylation used in the art for antibodies and antibody fragments(including, but not limited to, single domain antibodies and ScFvs);reference is made to, for example, Chapman, Nat. Biotechnol. 54, 531-545(2002); by Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456(2003); by Harris and Chess, Nat. Rev. Drug Discov. 2 (2003); and inWO04060965. Various reagents for pegylation of proteins are alsocommercially available, for example, from Nektar Therapeutics, USA.Preferably, site-directed pegylation is used, in particular, via acysteine-residue (see, for example, Yang et al., Protein Engineering 16(10):761-770 (2003). For example, for this purpose, PEG may be attachedto a cysteine residue that naturally occurs in a nanobody hereof. Ananobody hereof may be modified so as to suitably introduce one or morecysteine residues for attachment of PEG, or an amino acid sequencecomprising one or more cysteine residues for attachment of PEG may befused to the N- and/or C-terminus of a nanobody hereof, all usingtechniques of protein engineering known per se to the skilled person.Preferably, for the nanobodies and proteins hereof, a PEG is used with amolecular weight of more than 5000, such as more than 10,000 and lessthan 200,000, such as less than 100,000; for example, in the range of20,000-80,000. Another, usually less preferred modification comprisesN-linked or O-linked glycosylation, usually as part of co-translationaland/or post-translational modification, depending on the host cell usedfor expressing the nanobody or polypeptide hereof. Another technique forincreasing the half-life of a nanobody may comprise the engineering intobifunctional nanobodies (for example, one nanobody against the targetGPCR and one against a serum protein such as albumin) or into fusions ofnanobodies with peptides (for example, a peptide against a serum proteinsuch as albumin).

Yet another modification may comprise the introduction of one or moredetectable labels or other signal-generating groups or moieties,depending on the intended use of the labeled protein binding domain, inparticular, the nanobody. Suitable labels and techniques for attaching,using and detecting them will be clear to the skilled person, and, forexample, include, but are not limited to, fluorescent labels (such asfluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde, and fluorescamine and fluorescentmetals such as Eu or others metals from the lanthanide series),phosphorescent labels, chemiluminescent labels or bioluminescent labels(such as luminal, isoluminol, theromatic acridinium ester, imidazole,acridinium salts, oxalate ester, dioxetane or GFP and its analogs),radio-isotopes, metals, metals chelates or metallic cations or othermetals or metallic cations that are particularly suited for use in invivo, in vitro or in situ diagnosis and imaging, as well as chromophoresand enzymes (such as malate dehydrogenase, staphylococcal nuclease,delta-V-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase,asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease,catalase, glucose-VI-phosphate dehydrogenase, glucoamylase andacetylcholine esterase). Other suitable labels will be clear to theskilled person and, for example, include moieties that can be detectedusing NMR or ESR spectroscopy. Such labeled nanobodies and polypeptideshereof may, for example, be used for in vitro, in vivo or in situ assays(including immunoassays known per se such as ELISA, RIA, EIA and other“sandwich assays,” etc.) as well as in vivo diagnostic and imagingpurposes, depending on the choice of the specific label. As will beclear to the skilled person, another modification may involve theintroduction of a chelating group, for example to chelate one of themetals or metallic cations referred to above. Suitable chelating groups,for example, include, without limitation, diethylenetriaminepentaaceticacid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Yet anothermodification may comprise the introduction of a functional group that isone part of a specific binding pair, such as the biotin-(strept)avidinbinding pair. Such a functional group may be used to link the nanobodyhereof to another protein, polypeptide or chemical compound that isbound to the other half of the binding pair, i.e., through formation ofthe binding pair. For example, a nanobody hereof may be conjugated tobiotin, and linked to another protein, polypeptide, compound or carrierconjugated to avidin or streptavidin. For example, such a conjugatednanobody may be used as a reporter, for example, in a diagnostic systemwhere a detectable signal-producing agent is conjugated to avidin orstreptavidin. Such binding pairs may, for example, also be used to bindthe nanobody hereof to a carrier, including carriers suitable forpharmaceutical purposes. One non-limiting example is the liposomalformulations described by Cao and Suresh, Journal of Drug Targeting 8(4):257 (2000). Such binding pairs may also be used to link atherapeutically active agent to the nanobody hereof.

In a particular embodiment, the nanobody hereof is bivalent and formedby bonding together, either chemically or by recombinant DNA techniques,two monovalent single domains of heavy chains. In another particularembodiment, the nanobody hereof is bi-specific and formed by bondingtogether two variable domains of heavy chains, each with a differentspecificity. Similarly, polypeptides comprising multivalent ormulti-specific nanobodies are included here as non-limiting examples.Preferably, a monovalent nanobody hereof is such that it will bind to anextracellular part, region, domain, loop or other extracellular epitopeof a functional conformational state of a GPCR, more preferably, anactive conformational state of a GPCR, with an affinity less than 500nM, preferably less than 200 nM, more preferably less than 10 nM, suchas less than 500 pM. Alternatively, a monovalent nanobody hereof is suchthat it will bind to an intracellular part, region, domain, loop orother intracellular epitope of a functional conformational state of aGPCR, more preferably, an active conformational state of a GPCR with anaffinity less than 500 nM, preferably less than 200 nM, more preferablyless than 10 nM, such as less than 500 pM. Also, according to thisaspect, any multivalent or multispecific (as defined herein) nanobodyhereof may also be suitably directed against two or more differentextracellular or intracellular parts, regions, domains, loops or otherextracellular or intracellular epitopes on the same antigen, forexample, against two different extracellular or intracellular loops oragainst two different extracellular or intracellular parts of thetransmembrane domains. Such multivalent or multispecific nanobodieshereof may also have (or be engineered and/or selected for) increasedavidity and/or improved selectivity for the desired GPCR, and/or for anyother desired property or combination of desired properties that may beobtained by the use of such multivalent or multispecific nanobodies. Ina particular embodiment, such multivalent or multispecific nanobodieshereof may also have (or be engineered and/or selected for) improvedefficacy in modulating signaling activity of a GPCR (see also furtherherein). It will be appreciated that the multivalent or multispecificnanobodies hereof may additionally be suitably directed to a differentantigen, such as, but not limiting to, a ligand interacting with a GPCRor one or more downstream signaling proteins.

A second aspect hereof relates to a complex comprising (i) a proteinbinding domain hereof, (ii) a GPCR in a functional conformational state,and optionally, (iii) a receptor ligand. A “receptor ligand” or a“ligand,” as defined herein, may be a small compound, a protein, apeptide, a protein scaffold, a nucleic acid, an ion, a carbohydrate oran antibody, or any suitable fragment derived thereof, and the like.Preferentially, the ligand is from the agonist class and the receptor isin an active conformational state. The ligand may also be an inverseagonist, an antagonist, or a biased ligand. The ligands may beorthosteric or allosteric. Ligands also include allosteric modulators,potentiators, enhancers, negative allosteric modulators and inhibitors.They may have biological activity by themselves or they may modulateactivity only in the presence of another ligand. Neubig et al. (2003)describe many of the classes of ligands.

Provided for is a complex wherein the protein binding domain is bound tothe GPCR; preferably, the protein binding domain is bound to the GPCR,wherein the GPCR is bound to a receptor ligand. To illustrate thisfurther, and without the purpose of being limitative, a stable ternarycomplex containing a nanobody, a GPCR and an agonist can be purified byaffinity chromatography or gel filtration from a mixture, containing (1)a nanobody that is specific for the active conformation of that GPCR,(2) the detergent-solubilized receptor and (3) an agonist.

In another embodiment, the complex is crystalline. So, a crystal of thecomplex is also provided, as well as methods of making the crystal,which are described in greater detail below. A crystalline form of acomplex may comprise (i) a protein binding domain hereof, (ii) a GPCR ina functional conformational state, preferably an active conformationalstate, and optionally, (iii) a receptor ligand, is envisaged, whereinthe crystalline form is obtained by the use of a protein binding domainhereof.

In yet another embodiment, the complex hereof is in a solubilized form,for example, after aqueous solubilization with a detergent. In analternative preferred embodiment, the complex hereof is immobilized to asolid support. Non-limiting examples of solid supports, as well asmethods and techniques for immobilization, are described further in thedetailed description. In still another embodiment, the complex hereof isin a “cellular composition,” including an organism, a tissue, a cell, acell line, and a membrane composition derived from the organism, tissue,cell or cell line. The membrane composition is also meant to include anyliposomal composition that may comprise natural or synthetic lipids or acombination thereof. Examples of membrane or liposomal compositionsinclude, but are not limited to, organelles, membrane preparations,virus-like lipoparticles, lipid layers (bilayers and monolayers), lipidvesicles, high-density lipoparticles (e.g., nanodisks), and the like. Itwill be appreciated that a cellular composition, or a membrane-like orliposomal composition, may comprise natural or synthetic lipids.

Accordingly, a third aspect relates to a cellular composition, includinga membrane or liposomal composition derived thereof (all as definedhereinabove), comprising a protein binding domain and/or a complexhereof. Preferably, the cellular composition providing and/or expressingthe protein binding domain is capable of stabilizing and/or inducing afunctional conformational state of a GPCR upon binding of the proteinbinding domain. It will be understood that it is essential to retainsufficient functionality of the respective proteins, for which methodsand techniques are available and known to the person skilled in the art.It will also be appreciated that the cellular composition may provideand/or express a target GPCR endogenously or exogenously.

Preparations of GPCRs formed from membrane fragments ormembrane-detergent extracts are reviewed in detail in Cooper (2004),incorporated herein by reference. Non-limiting examples of solubilizedreceptor preparations are further provided in the Example section.

Transfection of target cells (e.g., mammalian cells) can be carried outfollowing principles outlined by Sambrook and Russel (Molecular Cloning,A Laboratory Manual, 3rd Edition, Volume 3, Chapter 16, Sections16.1-16.54). In addition, viral transduction can also be performed usingreagents such as adenoviral vectors. Selection of the appropriate viralvector system, regulatory regions and host cell is common knowledgewithin the level of ordinary skill in the art. The resulting transfectedcells are maintained in culture or frozen for later use according tostandard practices. Preferably, cells are eukaryotic cells, for example,yeast cells, or cultured cell lines, for example, mammalian cell lines,preferably human cell lines, that express a GPCR of interest. Preferredcell lines functionally expressing the protein binding domain and/or theGPCR include insect cells (e.g., SF-9), human cell lines (e.g., HEK293),rodent cell lines (e.g., CHO-K1), and camelid cell lines (Dubca).

The protein binding domains or the complexes or the cellular compositionas described hereinbefore can be used in a variety of context andapplications, which will be described in further detail below.

Crystallization and Resolving the Structure of a GPCR

Crystallization of membrane proteins including GPCRs remains aformidable challenge. Although expression and purification methods areappearing that allow for the generation of milligram quantities,achieving stability with these molecules is perhaps the most difficulthurdle to overcome. Purification necessitates a release of the GPCR fromthe lipid bilayer by detergent solubilization, a process during whichhydrophobic surfaces of the protein are coated with surfactant monomersto form a protein-detergent complex (PDC). However, the detergent beltformed around the protein is a poor replacement for the lipid bilayer,as much of the lateral pressure exerted on the protein by thesurrounding lipids is lost. Thus, solubilization of membrane proteinsoften results in destabilization, unfolding and subsequent aggregation.GPCRs other than rhodopsin typically have poor stability in detergentsand are prone to aggregation and proteolysis. Efforts to crystallizeGPCRs have been frustrated by other intrinsic characteristics ofintegral membrane proteins. The seven hydrophobic transmembrane helicesof GPCRs make poor surfaces for crystal contacts, and the extracellularand intracellular domains are often relatively short and/or poorlystructured. Besides for rhodopsin (an atypical GPCR in terms of naturalabundance and stability), the first crystals of GPCRs were obtained from02AR bound to a Fab fragment that recognized an epitope composed of theamino and carboxyl terminal ends of the third intracellular loopconnecting TMs 5 and 6 (Rasmussen, 2007). In the second approach, thethird intracellular loop was replaced by T4 lysozyme (β₂AR-T4L;Rosenbaum, 2007). Finally, the remarkable versatility of GPCRs assignaling molecules can be attributed to its flexible and dynamicthree-dimensional structure. Unfortunately, such dynamic behavior isparticularly challenging for high-resolution structure analysis. Growingdiffraction quality crystals requires stable, conformationallyhomogenous protein. As such, diffraction-quality crystals of a native,unbound GPCR are difficult to obtain and, even when this goal isachieved, the crystal structure will represent only one of the manynative conformations. Many of these problems can be solved in theinvention by the use of protein binding domains, in particular,nanobodies, as tools for stabilizing, purifying and crystallizingspecific conformational states of GPCRs for high-resolution structureanalysis.

It is thus one of the aims hereof to use protein binding domains astools to stabilize GPCR proteins and further to use these proteinbinding domains as co-crystallization aids for GPCRs, or in other words,to facilitate crystallogenesis of GPCRs, preferably in a functionalconformational state.

Accordingly, a fourth aspect relates to the use of a protein bindingdomain hereof, or in specific embodiments, a complex comprising theprotein binding domain or a cellular composition providing the proteinbinding domain, to stabilize a GPCR in a functional conformationalstate, in particular, in an active conformational state; and/or toinduce the formation of a particular functional (preferably, active)conformational state within a GPCR. It will be appreciated that such aprotein binding domain is a very useful tool to purify, to crystallizeand/or to solve the structure of a GPCR in a functional conformationalstate, in particular, in its active conformational state. As clearlyoutlined hereinbefore, it should be clear that the protein bindingdomains hereof invention, which are to be used for purifying,stabilizing, crystallizing and/or solving the structure of a GPCR, maybe directed against any desired GPCR and may specifically bind to orrecognize a conformational epitope of a functional conformational state,preferably an active conformational state, of any desired GPCR. Inparticular, the conformational epitope can be part of an intracellularor extracellular region, or an intramembranous region, or a domain orloop structure of any desired GPCR.

First, protein binding domains hereof may increase the thermostabilityof detergent-solubilized receptors, stabilized in a particularconformational state, protecting them from proteolytic degradationand/or aggregation and facilitating the purification and concentrationof homogeneous samples of correctly folded receptor. Persons of ordinaryskill in the art will recognize that such samples are the preferredstarting point for the generation of diffracting crystals.

The crystallization of the purified receptor is another major bottleneckin the process of macromolecular structure determination by X-raycrystallography. Successful crystallization requires the formation ofnuclei and their subsequent growth to crystals of suitable size. Crystalgrowth generally occurs spontaneously in a supersaturated solution as aresult of homogenous nucleation. Proteins may be crystallized in atypical sparse matrix screening experiment, in which precipitants,additives and protein concentration are sampled extensively, andsupersaturation conditions suitable for nucleation and crystal growthcan be identified for a particular protein. Related to the sparse matrixscreening approach is to generate structural variation in the proteinitself, for example, by adding ligands that bind the protein, or bymaking different mutations, preferentially in surface residues of thetarget protein or by trying to crystallize different species orthologuesof the target protein (Chang 1998). One unexpected finding is theusefulness of protein binding domains, such as nanobodies, thatspecifically bind to a GPCR to introduce a degree of structuralvariation upon binding while preserving the overall fold of the GPCR.Different nanobodies will generate different quaternary structuresproviding new distinct interfaces for crystal lattice formationresulting in multiple crystal forms while preserving the overall fold ofthe GPCR.

Because crystallization involves an unfavorable loss of conformationalentropy in the molecule to be assembled in the crystal lattice, methodsthat reduce the conformational entropy of the target while still insolution should enhance the likelihood of crystallization by loweringthe net entropic penalty of lattice formation. The “surface entropyreduction” approach has proved to be highly effective (Derewenda 2004).Likewise, binding partners such as ions, small molecule ligands, andpeptides can reduce the conformational heterogeneity by binding to andstabilizing a subset of conformational states of a protein. Althoughsuch binding partners are effective, not all proteins have a knownbinding partner, and even when a binding partner is known, its affinity,solubility, and chemical stability may not be compatible withcrystallization trials. Therefore, it was surprisingly found that theprotein binding domains hereof, in particular, the nanobodies, can beused as tools to increase the probability of obtaining well-orderedcrystals by minimizing the conformational heterogeneity in the targetGPCR by binding to a particular conformation of the receptor.

Crystallization of GPCRs for high-resolution structural studies isparticularly difficult because of the amphipathic surface of thesemembrane proteins. Embedded in the membrane bilayer, the contact sitesof the protein with the acyl chains of the phospholipids arehydrophobic, whereas the polar surfaces are exposed to the polar headgroups of the lipids and to the aqueous phases. To obtain well-orderedthree-dimensional crystals—a prerequisite to X-ray structural analysisat high resolution—GPCRs are solubilized with the help of detergents andpurified as protein-detergent complexes. The detergent micelle coversthe hydrophobic surface of the membrane protein in a belt-like manner(Hunte and Michel 2002; Ostermeier et al. 1995). GPCR-detergentcomplexes form three-dimensional crystals in which contacts betweenadjacent protein molecules are made by the polar surfaces of the proteinprotruding from the detergent micelle (Day et al. 2007). Obviously, thedetergent micelle requires space in the crystal lattice. Althoughattractive interactions between the micelles might stabilize the crystalpacking (Rasmussen et al. 2007; Dunn et al. 1997), these interactions donot lead to rigid crystal contacts. Because many membrane proteins,including GPCRs, contain relatively small or highly flexible hydrophilicdomains, a strategy to increase the probability of getting well-orderedcrystals is to enlarge the polar surface of the protein and/or to reducetheir flexibility. In order for the nanobodies hereof to be used toenlarge the polar surfaces of the protein, supplementing the amount ofprotein surface can facilitate primary contacts between molecules in thecrystal lattice. Nanobodies hereof can also reduce the flexibility ofits extracellular regions to grow well-ordered crystals. Nanobodies areespecially suited for this purpose because they are composed of onesingle rigid globular domain and are devoid of flexible linker regionsunlike conventional antibodies or fragments derived such as Fabs.

In a further embodiment, the complex comprising the protein bindingdomain hereof and the target GPCR in a functional conformational state,preferably an active conformational state, may be crystallized using anyof a variety of specialized crystallization methods for membraneproteins, many of which are reviewed in Caffrey (2003). In generalterms, the methods are lipid-based methods that include adding lipid tothe GPCR-nanobody complex prior to crystallization. Such methods havepreviously been used to crystallize other membrane proteins. Many ofthese methods, including the lipidic cubic phase crystallization methodand the bicelle crystallization method, exploit the spontaneousself-assembling properties of lipids and detergent as vesicles(vesicle-fusion method), discoidal micelles (bicelle method), and liquidcrystals or mesophases (in meso or cubic-phase method). Lipidic cubicphase crystallization methods are described in, for example, Landau etal. 1996; Gouaux 1998; Rummel et al. 1998; Nollert et al. 2004, whichpublications are incorporated by reference for disclosure of thosemethods. Bicelle crystallization methods are described in, for example,Faham et al. 2005; Faham et al. 2002, which publications areincorporated by reference for disclosure of those methods.

Further encompassed is the use of a protein binding domain as describedhereinbefore to solve a structure of a GPCR. The structure of a protein,in particular a GPCR, includes the primary, secondary, tertiary and, ifapplicable, quaternary structure of the protein. “Solving the structure”as used herein refers to determining the arrangement of atoms or theatomic coordinates of a protein, and is often done by a biophysicalmethod, such as X-ray crystallography.

In x-ray crystallography, the diffraction data when properly assembledgives the amplitude of the 3D Fourier transform of the molecule'selectron density in the unit cell. If the phases are known, the electrondensity can be simply obtained by Fourier synthesis. For a proteincomplex, the success to derive phase information from molecularreplacement (MR) alone is questionable when the fraction of proteinswith a known structure (the search models) is low (less than 50% of theamino acid content) and/or when the crystals exhibit limited diffractionquality. While the combination of multiple isomorphous replacement (MIR)and MR phasing has proven successful for protein complexes (e.g.,Ostermeier et al. 1995; Li et al. 1997; Hunte et al. 2000), therequirement of producing a good heavy atom derivative is almost alwaysproblematic. Over the past decade, classical MIR approaches havegenerally been superseded by the use of anomalous dispersion dataprincipally using selenomethionine (SeMet) incorporation (MAD or SAD)(Hendrickson 1991). In fact, the anomalous experimental data usingSe-edge energies generally provide superior and less biased phaseinformation compared with either MIR or model-based MR phasing data. Oneembodiment relates to the use of nanobodies for the phasing ofGPCR-nanobody complexes by MR or MAD. Nanobodies generally expressrobustly and are suitable for SeMet incorporation. Phasing aGPCR-nanobody complex by introducing all the SeMet sites in the nanobodyalone circumvents the need to incorporate SeMet sites in the GPCR.

In many cases, obtaining a diffraction-quality crystal is the chiefbarrier to solving its atomic-resolution structure. Thus, according tospecific embodiments, the protein binding domains as describedhereinbefore, in particular, the nanobodies, can be used to improve thediffraction quality of the crystals so that the GPCR protein crystalstructure can be solved.

There is great interest in structural information to help guide GPCRdrug discovery. For the GPCRs whose structures have now been solved,these modeling efforts have been shown to be imprecise at the levelrequired by in silico drug designers. With the inactive-state structuresof β₂AR, the β₁AR and the A2A receptor, pharmaceutical chemists now haveexperimental data to guide the development of ligands for several activetherapeutic targets. However, the value of these high-resolutionstructures for in silico screening may be limited. Recent moleculardocking studies using the β₂AR crystal structure as a templateidentified six new β₂AR ligands that bind with affinities ranging from 9nM to 4 μM; however, every compound exhibited inverse agonist activity.Beyond the crystallization of more GPCRs, methods for acquiringstructures of receptors bound to different classes of ligands includingagonists, antagonists, allosteric regulators and/or G proteins must bedeveloped. For example, agonist-bound receptor crystals may providethree-dimensional representations of the active states of GPCRs. Thesestructures will help clarify the conformational changes connecting theligand-binding and G-protein-interaction sites, and lead to more precisemechanistic hypotheses and eventually new therapeutics. Given theconformational flexibility inherent to ligand-activated GPCRs and thegreater heterogeneity exhibited by agonist-bound receptors, stabilizingsuch a state is not easy. Such efforts can benefit from thestabilization of the agonist-bound receptor conformation by the additionof protein binding domains that are specific for an activeconformational state of the receptor. Especially suited are nanobodiesthat show G-protein-like behavior and exhibit cooperative propertieswith respect to agonist binding, as are provided herein (see Examplesection).

Accordingly, also provided is a method of determining a crystalstructure of a GPCR in a functional conformational state, the methodcomprising the steps of:

-   -   (i) providing a protein binding domain hereof, a target GPCR,        and optionally a receptor ligand,    -   (ii) forming a complex of the protein binding domain, the GPCR,        and optionally the receptor ligand, and    -   (iii) crystallizing the complex of step (ii) to form a crystal,    -   wherein the crystal structure is determined of a GPCR in a        functional conformational state, preferably the active        conformational state.

Determining the crystal structure may be done by a biophysical methodsuch as X-ray crystallography. The method may further comprises a stepof obtaining the atomic coordinates of the crystal (as definedhereinbefore).

Capturing and/or Purifying a GPCR in a Functional Conformational State

In yet another embodiment, provided is a method for capturing and/orpurifying a GPCR in a functional conformational state, preferably anactive conformational state, by making use of any of the above-describedprotein binding domains, or complexes or cellular compositionscomprising such protein binding domains. Capturing and/or purifying aGPCR in a functional conformational state, preferably an activeconformational state, hereof, will allow subsequent crystallization,ligand characterization and compound screening, and immunizations,amongst others. In practice, such methods and techniques include,without limitation, affinity-based methods such as affinitychromatography, affinity purification, immunoprecipitation, proteindetection, immunochemistry, and surface-display, amongst others, and areall well known by the skilled in the art.

Thus, described is the use of a protein binding domain hereof, or acomplex or a cellular composition comprising the same as describedhereinbefore, to capture a GPCR in a functional conformational state,preferably to capture a GPCR in its active conformational state.Optionally, but not necessarily, capturing of a GPCR in its functionalconformational state as described above may include capturing a GPCR ina functional conformational state in complex with a receptor ligand orone or more downstream interacting proteins.

Accordingly, also provided is a method of capturing a GPCR in afunctional conformational state, the method comprising the steps of:

-   -   (i) providing a protein binding domain hereof and a target GPCR,        and,    -   (ii) forming a complex of the protein binding domain and the        GPCR,    -   wherein a GPCR is captured in a functional conformational state,        preferably an active conformational state.

More specifically, also provided is a method of capturing a GPCR in afunctional conformational state, the method comprising the steps of:

-   -   (i) applying a solution containing a GPCR in a plurality of        conformational states to a solid support possessing an        immobilized protein binding domain hereof,    -   (ii) forming a complex of the protein binding domain and the        GPCR, and    -   (iii) removing weakly bound or unbound molecules,    -   wherein a GPCR is captured in a functional conformational state,        preferably an active conformational state.

It will be appreciated that any of the methods as described above mayfurther comprise the step of purifying the complex of the proteinbinding domain and the GPCR in its functional conformational state.

Therapeutic and Diagnostic Applications

Traditionally, small molecules are used for development of drugsdirected against GPCRs, not only because pharmaceutical companies havehistorical reasons to work with these, but, more importantly, because ofthe structural constraints of Family 1 GPCRs, which have the ligandbinding site within the transmembrane cleft (Nat. Rev. Drug Discov.(2004), the state of GPCR research in 2004, Nature Reviews DrugDiscovery GPCR Questionnaire Participants 3(7):575, 577-626). For thisreason, it proved to be difficult or impossible to generate monoclonalantibodies against this target class. The protein binding domainshereof, in particular, the nanobodies, can solve this particular problemby means of their intrinsic property of binding via extended CDR loopsinto cavities.

Accordingly, a fifth aspect hereof relates to a pharmaceuticalcomposition comprising a therapeutically effective amount of a proteinbinding domain hereof and at least one of a pharmaceutically acceptablecarrier, adjuvant or diluent.

A “carrier” or “adjuvant,” in particular, a “pharmaceutically acceptablecarrier” or “pharmaceutically acceptable adjuvant” is any suitableexcipient, diluent, carrier and/or adjuvant that, by themselves, do notinduce the production of antibodies harmful to the individual receivingthe composition nor do they elicit protection. So, pharmaceuticallyacceptable carriers are inherently non-toxic and nontherapeutic, andthey are known to the person skilled in the art. Suitable carriers oradjuvantia typically comprise one or more of the compounds included inthe following non-exhaustive list: large slowly metabolizedmacromolecules such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers andinactive virus particles. Carriers or adjuvants may be, as anon-limiting example, Ringer's solution, dextrose solution or Hank'ssolution. Non-aqueous solutions such as fixed oils and ethyl oleate mayalso be used. A preferred excipient is 5% dextrose in saline. Theexcipient may contain minor amounts of additives such as substances thatenhance isotonicity and chemical stability, including buffers andpreservatives.

The administration of a protein binding domain hereof or apharmaceutical composition thereof may be by way of oral, inhaled orparenteral administration. In particular embodiments, the proteinbinding domain is delivered through intrathecal orintracerebroventricular administration. The active compound may beadministered alone or preferably formulated as a pharmaceuticalcomposition. An amount effective to treat a certain disease or disorderthat express the antigen recognized by the protein binding domaindepends on the usual factors such as the nature and severity of thedisorder being treated and the weight of the mammal. However, a unitdose will normally be in the range of 0.1 mg to 1 g, for example, to 0.1to 500 mg, for example, 0.1 to 50 mg, or 0.1 to 2 mg of protein bindingdomain or a pharmaceutical composition thereof. Unit doses will normallybe administered once a month, once a week, bi-weekly, once or more thanonce a day, for example, two, three, or four times a day, more usuallyone to three times a day. It is greatly preferred that the proteinbinding domain or a pharmaceutical composition thereof is administeredin the form of a unit-dose composition, such as a unit dose oral,parenteral, or inhaled composition. Such compositions are prepared byadmixture and are suitably adapted for oral, inhaled or parenteraladministration, and as such, may be in the form of tablets, capsules,oral liquid preparations, powders, granules, lozenges, reconstitutablepowders, injectable and infusable solutions or suspensions orsuppositories or aerosols. Tablets and capsules for oral administrationare usually presented in a unit dose, and contain conventionalexcipients such as binding agents, fillers, diluents, tableting agents,lubricants, disintegrants, colorants, flavorings, and wetting agents.The tablets may be coated according to well-known methods in the art.Suitable fillers for use include cellulose, mannitol, lactose and othersimilar agents. Suitable disintegrants include starch,polyvinylpyrrolidone and starch derivatives such as sodium starchglycolate. Suitable lubricants include, for example, magnesium stearate.Suitable pharmaceutically acceptable wetting agents include sodiumlauryl sulphate. These solid oral compositions may be prepared byconventional methods of blending, filling, tableting, or the like.Repeated blending operations may be used to distribute the active agentthroughout those compositions employing large quantities of fillers.Such operations are, of course, conventional in the art. Oral liquidpreparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups, or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents (for example, sorbitol,syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminium stearate gel or hydrogenated edible fats),emulsifying agents (for example, lecithin, sorbitan monooleate, oracacia), non-aqueous vehicles (which may include edible oils, forexample, almond oil, fractionated coconut oil, oily esters such asesters of glycerine, propylene glycol, or ethyl alcohol), preservatives(for example, methyl or propyl p-hydroxybenzoate or sorbic acid), and,if desired, conventional flavoring or coloring agents. Oral formulationsalso include conventional sustained release formulations, such astablets or granules having an enteric coating. Preferably, compositionsfor inhalation are presented for administration to the respiratory tractas a snuff or an aerosol or solution for a nebulizer, or as a microfinepowder for insufflation, alone or in combination with an inert carriersuch as lactose. In such a case, the particles of active compoundsuitably have diameters of less than 50 microns, preferably less than 10microns, for example between 1 and 5 microns, such as between 2 and 5microns. A favored inhaled dose will be in the range of 0.05 to 2 mg,for example, 0.05 to 0.5 mg, 0.1 to 1 mg or 0.5 to 2 mg. For parenteraladministration, fluid unit dose forms are prepared containing a compoundhereof and a sterile vehicle. The active compound, depending on thevehicle and the concentration, can be either suspended or dissolved.Parenteral solutions are normally prepared by dissolving the compound ina vehicle and filter sterilizing before filling into a suitable vial orampoule and sealing. Advantageously, adjuvants such as a localanesthetic, preservatives and buffering agents are also dissolved in thevehicle. To enhance the stability, the composition can be frozen afterfilling into the vial and the water removed under vacuum. Parenteralsuspensions are prepared in substantially the same manner except thatthe compound is suspended in the vehicle instead of being dissolved andsterilized by exposure to ethylene oxide before suspending in thesterile vehicle. Advantageously, a surfactant or wetting agent isincluded in the composition to facilitate uniform distribution of theactive compound. Where appropriate, small amounts of bronchodilators,for example, sympathomimetic amines (such as isoprenaline, isoetharine,salbutamol, phenylephrine and ephedrine), xanthine derivatives (such astheophylline and aminophylline), corticosteroids (such as prednisolone),and adrenal stimulants (such as ACTH) may be included. As is commonpractice, the compositions will usually be accompanied by written orprinted directions for use in the medical treatment concerned.

Delivery of protein binding domains, in particular nanobodies, intocells may be performed as described for peptides, polypeptides andproteins. If the antigen is extracellular or an extracellular domain,the protein binding domain may exert its function by binding to thisdomain, without need for intracellular delivery. The protein bindingdomains hereof as described herein may target intracellularconformational epitopes of GPCRs of interest. To use these proteinbinding domains as effective and safe therapeutics inside a cell,intracellular delivery may be enhanced by protein transduction ordelivery systems known in the art. Protein transduction domains (PTDs)have attracted considerable interest in the drug delivery field fortheir ability to translocate across biological membranes. The PTDs arerelatively short (one- to 35-amino acid) sequences that confer thisapparent translocation activity to proteins and other macromolecularcargo to which they are conjugated, complexed or fused (Sawant andTorchilin 2010). The HIV-derived TAT peptide (YGRKKRRQRRR (SEQ IDNO:71), for example, has been used widely for intracellular delivery ofvarious agents ranging from small molecules to proteins, peptides, rangeof pharmaceutical nanocarriers and imaging agents. Alternatively,receptor-mediated endocytic mechanisms can also be used forintracellular drug delivery. For example, the transferrinreceptor-mediated internalization pathway is an efficient cellularuptake pathway that has been exploited for site-specific delivery ofdrugs and proteins (Qian et al. 2002). This is achieved eitherchemically by conjugation of transferrin with therapeutic drugs orproteins or genetically by infusion of therapeutic peptides or proteinsinto the structure of transferrin. Naturally existing proteins (such asthe iron-binding protein transferrin) are very useful in this area ofdrug targeting since these proteins are biodegradable, nontoxic, andnon-immunogenic. Moreover, they can achieve site-specific targeting dueto the high amounts of their receptors present on the cell surface.Still other delivery systems include, without the purpose of beinglimitative, polymer- and liposome-based delivery systems.

The efficacy of the protein binding domains hereof, and of compositionscomprising the same, can be tested using any suitable in vitro assay,cell-based assay, in vivo assay and/or animal model known per se, or anycombination thereof, depending on the specific disease or disorderinvolved.

A sixth aspect hereof relates to the use of the protein binding domainor the pharmaceutical composition as described hereinbefore to modulateGPCR signaling activity.

The protein binding domains hereof as described herein may bind to theGPCR so as to activate or increase receptor signaling, or,alternatively, so as to decrease or inhibit receptor signaling. Theprotein binding domains hereof may also bind to the GPCR in such a waythat they block off the constitutive activity of the GPCR. The proteinbinding domains hereof may also bind to the GPCR in such a way that theymediate allosteric modulation (e.g., bind to the GPCR at an allostericsite). In this way, the protein binding domains hereof may modulate thereceptor function by binding to different regions in the receptor (e.g.,at allosteric sites). Reference is made, for example, to George et al.(2002), Kenakin (2002) and Rios et al. (2001). The protein bindingdomains hereof may also bind to the GPCR in such a way that they prolongthe duration of the GPCR-mediated signaling or that they enhancereceptor signaling by increasing receptor-ligand affinity. Further, theprotein binding domains hereof may also bind to the GPCR in such a waythat they inhibit or enhance the assembly of GPCR functional homomers orheteromers.

In one particular embodiment, the protein binding domain or thepharmaceutical composition as described hereinbefore blocksG-protein-mediated signaling.

In another embodiment, also envisaged is the protein binding domain orthe pharmaceutical composition as described hereinbefore for use in thetreatment of a GPCR-related disease, such as cancer, autoimmune disease,infectious disease, neurological disease, and cardiovascular disease.

Certain of the above-described protein binding domains may havetherapeutic utility and may be administered to a subject having acondition in order to treat the subject for the condition. Thetherapeutic utility for a protein binding domain may be determined bythe GPCR to which the protein binding domain binds in that signaling viathat GPCR is linked to the condition. In certain cases, the GPCR may beactivated in the condition by binding to a ligand. In other embodiments,the GPCR may be mutated to make it constitutively active, for example. Asubject protein binding domain may be employed for the treatment of aGPCR-mediated condition such as schizophrenia, migraine headache,reflux, asthma, bronchospasm, prostatic hypertrophy, ulcers, epilepsy,angina, allergy, rhinitis, cancer, e.g., prostate cancer, glaucoma andstroke. Further exemplary GPCR-related conditions at the On-lineMendelian Inheritance in Man database can be found at the world widewebsite of the NCBI. A particular embodiment hereof also envisions theuse of a protein binding domain or of a pharmaceutical composition forthe treatment of a GPCR-related disease or disorder.

In a more specific embodiment, the protein binding domain may bind tothe β₂-adrenergic receptor, in which case, it may be employed in thetreatment of a condition requiring relaxation of smooth muscle of theuterus or vascular system. Such a protein binding domain may be thusused for the prevention or alleviation of premature labor pains inpregnancy, or in the treatment of chronic or acute asthma, urticaria,psoriasis, rhinitis, allergic conjunctivitis, acinitis, hay fever, ormastocytosis, which conditions have been linked to theβ₂-adrenoreceptor. In these embodiments, the protein binding domain maybe employed as co-therapeutic agents for use in combination with otherdrug substances such as anti-inflammatory, bronchodilatory orantihistamine drug substances, particularly in the treatment ofobstructive or inflammatory airway diseases such as those mentionedhereinbefore, for example, as potentiators of therapeutic activity ofsuch drugs or as a means of reducing required dosaging or potential sideeffects of such drugs. A subject protein binding domain may be mixedwith the other drug substance in a fixed pharmaceutical composition orit may be administered separately, before, simultaneously with or afterthe other drug substance. In general terms, these protocols involveadministering to an individual suffering from a GPCR-related disease ordisorder an effective amount of a protein binding domain that modulatesa GPCR to modulate the GPCR in the host and treat the individual for thedisorder.

In some embodiments, where a reduction in activity of a certain GPCR isdesired, one or more compounds that decrease the activity of the GPCRmay be administered, whereas when an increase in activity of a certainGPCR is desired, one or more compounds that increase the activity of theGPCR activity may be administered.

A variety of individuals are treatable according to the subject methods.Generally, such individuals are mammals or mammalian, where these termsare used broadly to describe organisms that are within the classmammalia, including the orders carnivore (e.g., dogs and cats), rodentia(e.g., mice, guinea pigs, and rats), and primates (e.g., humans,chimpanzees, and monkeys). In many embodiments, the individuals will behumans. Subject treatment methods are typically performed on individualswith such disorders or on individuals with a desire to avoid suchdisorders.

According to still another embodiment, the protein binding domain or thecomplex hereof may also be useful for the diagnosis or prognosis of aGPCR-related disease, such as cancer, autoimmune disease, infectiousdisease, neurological disease, or cardiovascular disease.

Identification of Compounds Selectively Targeting a GPCR in a FunctionalConformational State

In the process of compound screening, lead optimization and drugdiscovery, there is a requirement for faster, more effective, lessexpensive and especially information-rich screening assays that providesimultaneous information on various compound characteristics and theireffects on various cellular pathways (i.e., efficacy, specificity,toxicity and drug metabolism). Thus, there is a need to quickly andinexpensively screen large numbers of compounds in order to identify newspecific ligands of a GPCR of interest, preferably conformation-specificligands, which may be potential new drug candidates. The presentinvention solves this problem by providing protein binding domains thatstabilize and/or lock a GPCR in a functional conformational state,preferably an active conformational state, that can then be used asimmunogens or selection reagents for screening in a variety of contexts.A major advantage of the protein binding domains hereof is that a GPCRcan be kept in a stabilized functional conformation, preferably in anactive state conformation. For example, library compounds thatselectively bind this active conformation of the receptor have a higherpropensity to behave as agonists because orthosteric or allostericstabilization of the active conformation of the GPCR elicits biologicalresponses. Another advantage is that the protein binding domainincreases the thermostability of the active conformation of the GPCR,thus protecting the GPCR against irreversible or thermal denaturationinduced by the non-native conditions used in compound screening and drugdiscovery, without the need to rely on mutant GPCRs with increasedstability. Another major advantage of the conformation-selective proteinbinding domains hereof is that they quickly and reliably screen for anddifferentiate between receptor agonists, inverse agonists, antagonistsand/or modulators as well as inhibitors of GPCRs and GPCR-dependentpathways, thereby increasing the likelihood of identifying a ligand withthe desired pharmacological properties.

To illustrate this further, it is a well-established concept that mostGPCRs exhibit higher agonist binding affinity when complexed with Gprotein. This is attributed to the cooperative interaction betweenagonist-occupied receptor and G protein (Delean et al. 1980). Theprotein binding domains hereof, in particular, the nanobodies, arecapable of stabilizing an active conformational state of a GPCR (andthus show G-protein-like behavior) and destabilizing inactiveconformational states, thus increasing the affinity of the GPCR foragonists and decreasing the affinity for inverse agonists orantagonists. It follows that the protein binding domains, such asnanobodies, hereof invention, that recognize the active functionalconformation of the GPCR can be efficiently used in high-throughputscreening assays to screen for agonists because they increase theaffinity of the receptor for agonists, relative to inverse agonists orantagonists. Reciprocally, protein binding domains, in particular,nanobodies, that stabilize the inactive state conformation of a GPCRwill increase the affinity for an inverse agonist or an antagonist anddecrease the affinity for an agonist. Such protein binding domains maybe used, for example, to screen for inverse agonists. Thus, proteinbinding domains, particularly nanobodies, that recognize particularfunctional conformational states, thus modulating the affinities foragonists and inverse agonists in a reciprocal way, also form parthereof.

Thus, according to a preferred embodiment, encompassed is the use of theprotein binding domains, or the complexes, or the cellular compositions,all as described hereinbefore, in screening and/or identificationprograms for conformation-specific binding partners of a GPCR, whichultimately might lead to potential new drug candidates.

According to one embodiment, envisaged is a method of identifyingcompounds capable of selectively binding to a functional conformationalstate of a GPCR, the method comprising the steps of:

-   -   (i) Providing a GPCR and a protein binding domain capable of        specifically binding to a functional conformational state of the        GPCR hereof,    -   (ii) Providing a test compound,    -   (iii) Evaluating whether the test compound binds to the        functional conformational state of the GPCR, and    -   (iv) Selecting a compound that selectively binds to the        functional conformational state of the GPCR.

Preferably, the above method further comprises a step of forming acomplex comprising the protein binding domain and the GPCR in afunctional conformational state, more preferably, in an activeconformational state.

Thus, the invention also envisages a method of identifying compoundscapable of selectively binding to a functional conformational state of aGPCR, the method comprising the steps of:

-   -   (i) Providing a complex comprising a protein binding domain        hereof and a GPCR in a functional conformational state,    -   (ii) Providing a test compound,    -   (iii) Evaluating whether the test compound binds to the        functional conformational state of the GPCR, and    -   (iv) Selecting a compound that binds to the functional        conformational state of the GPCR.

Preferably, the protein binding domain as used in any of the abovemethods is capable of stabilizing and/or inducing a functionalconformational state of a GPCR upon binding. Preferably, the functionalconformational state of a GPCR is selected from the group consisting ofa basal conformational state, or an active conformational state or aninactive conformational state (as defined hereinbefore). Mostpreferably, the functional conformational state of a GPCR is an activeconformational state.

In one other preferred embodiment, the protein binding domain as used inany of the above screening methods comprises an amino acid sequence thatcomprises four framework regions and three complementarity-determiningregions, or any suitable fragment thereof. Preferably, the proteinbinding domain is derived from a camelid antibody. More preferably, theprotein binding domain comprises a nanobody sequence, or any suitablefragment thereof. In particular, the nanobody comprises a sequenceselected from the group consisting of SEQ ID NOS: 1-29, or any suitablefragment thereof.

Other preferences for the protein binding domains and/or the complexesare as defined above with respect to the first and second aspect hereof.

In a preferred embodiment, the protein binding domain, the GPCR or thecomplex comprising the protein binding domain and the GPCR, as used inany of the above screening methods, are provided as whole cells, or cell(organelle) extracts such as membrane extracts or fractions thereof, ormay be incorporated in lipid layers or vesicles (comprising naturaland/or synthetic lipids), high-density lipoparticles, or anynanoparticle, such as nanodisks, or are provided as VLPs, so thatsufficient functionality of the respective proteins is retained.Preparations of GPCRs formed from membrane fragments ormembrane-detergent extracts are reviewed in detail in Cooper (2004),incorporated herein by reference. Alternatively, GPCRs and/or thecomplex may also be solubilized in detergents. Non-limiting examples ofsolubilized receptor preparations are further provided in the Examplesection.

Often, high-throughput screening of GPCR targets forconformation-specific binding partners will be preferred. This will befacilitated by immobilization of a protein binding domain hereof, a GPCRin a functional conformational state or a complex comprising them, ontoa suitable solid surface or support that can be arrayed or otherwisemultiplexed. Non-limiting examples of suitable solid supports includebeads, columns, slides, chips or plates.

More particularly, the solid supports may be particulate (e.g., beads orgranules, generally used in extraction columns) or in sheet form (e.g.,membranes or filters, glass or plastic slides, microtiter assay plates,dipstick, capillary fill devices or the like), which can be flat,pleated, or hollow fibers or tubes. The following matrices are given asexamples and are not exhaustive. Such examples could include silica(porous amorphous silica), i.e., the FLASH series of cartridgescontaining 60A irregular silica (32-63 μm or 35-70 μm) supplied byBiotage (a division of Dyax Corp.), agarose or polyacrylamide supports,for example, the Sepharose range of products supplied by AmershamPharmacia Biotech, or the Affi-Gel supports supplied by Bio-Rad. Inaddition, there are macroporous polymers, such as the pressure-stableAffi-Prep supports as supplied by Bio-Rad. Other supports that could beutilized include dextran, collagen, polystyrene, methacrylate, calciumalginate, controlled pore glass, aluminium, titanium and porousceramics. Alternatively, the solid surface may comprise part of amass-dependent sensor, for example, a surface plasmon resonancedetector. Further examples of commercially available supports arediscussed in, for example, Protein Immobilization, R.F. Taylor ed.,Marcel Dekker, Inc., New York, (1991).

Immobilization may be either non-covalent or covalent. In particular,non-covalent immobilization or adsorption on a solid surface of theprotein binding domain, the GPCR or the complex comprising the proteinbinding domain and the GPCR, hereof invention, may occur via a surfacecoating with any of an antibody, or streptavidin or avidin, or a metalion, recognizing a molecular tag attached to the protein binding domainor the GPCR, according to standard techniques known by the skilledperson (e.g., biotin tag, Histidine tag, etc.).

In particular, the protein binding domain, the GPCR or the complexcomprising the protein binding domain and the GPCR, hereof, may beattached to a solid surface by covalent cross-linking using conventionalcoupling chemistries. A solid surface may naturally comprisecross-linkable residues suitable for covalent attachment or it may becoated or derivatized to introduce suitable cross-linkable groupsaccording to methods well known in the art. In one particularembodiment, sufficient functionality of the immobilized protein isretained following direct covalent coupling to the desired matrix via areactive moiety that does not contain a chemical spacer arm. Furtherexamples and more detailed information on immobilization methods ofantibody (fragments) on solid supports are discussed in Jung et al.(2008); similarly, membrane receptor immobilization methods are reviewedin Cooper (2004); both herein incorporated by reference.

Advances in molecular biology, particularly through site-directedmutagenesis, enable the mutation of specific amino acid residues in aprotein sequence. The mutation of a particular amino acid (in a proteinwith known or inferred structure) to a lysine or cysteine (or otherdesired amino acid) can provide a specific site for covalent coupling,for example. It is also possible to reengineer a specific protein toalter the distribution of surface available amino acids involved in thechemical coupling (Kallwass et al. 1993), in effect controlling theorientation of the coupled protein. A similar approach can be applied tothe protein binding domains hereof, as well as to the conformationallystabilized GPCRs, whether or not comprised in a complex, so as toprovide a means of oriented immobilization without the addition of otherpeptide tails or domains containing either natural or unnatural aminoacids. In case of an antibody or an antibody fragment, such as ananobody, introduction of mutations in the framework region ispreferred, minimizing disruption to the antigen-binding activity of theantibody (fragment).

Conveniently, the immobilized proteins may be used in immunoadsorptionprocesses such as immunoassays, for example, ELISA, or immunoaffinitypurification processes by contacting the immobilized proteins hereofwith a test sample according to standard methods conventional in theart. Alternatively, and particularly for high-throughput purposes, theimmobilized proteins can be arrayed or otherwise multiplexed.Preferably, the immobilized proteins hereof are used for the screeningand selection of compounds that specifically bind to a GPCR in afunctional conformational state, in particular, a GPCR in an activeconformational state.

It will be appreciated that either the protein binding domain or theGPCR in a functional conformational state, or the complex comprising theprotein binding domain and the GPCR, may be immobilized, depending onthe type of application or the type of screening that needs to be done.Also, the choice of the GPCR-stabilizing protein binding domain(targeting a particular conformational epitope of the GPCR), willdetermine the orientation of the GPCR and, accordingly, the desiredoutcome of the compound identification, e.g., compounds specificallybinding to extracellular parts, intramembranal parts or intracellularparts of the conformationally stabilized GPCR.

In an alternative embodiment, the test compound (or a library of testcompounds) may be immobilized on a solid surface, such as a chipsurface, whereas the protein binding domain, the GPCR or the complex areprovided, for example, in a detergent solution or in a membrane-likepreparation.

Most preferably, neither the protein binding domain, nor the GPCR, northe test compound is immobilized, for example, in phage-displayselection protocols in solution, or radioligand binding assays.

Various methods may be used to determine binding between the stabilizedGPCR and a test compound, including, for example, enzyme-linkedimmunosorbent assays (ELISA), surface Plasmon resonance assays,chip-based assays, immunocytofluorescence, yeast two-hybrid technologyand phage display, which are common practice in the art, for example, inSambrook et al. (2001), Molecular Cloning, A Laboratory Manual, ThirdEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Other methods of detecting binding between a test compound and a GPCRinclude ultrafiltration with ion spray mass spectroscopy/HPLC methods orother (bio)physical and analytical methods. Fluorescence EnergyResonance Transfer (FRET) methods, for example, well known to thoseskilled in the art, may also be used. It will be appreciated that abound test compound can be detected using a unique label or tagassociated with the compound, such as a peptide label, a nucleic acidlabel, a chemical label, a fluorescent label, or a radio frequency tag,as described further herein.

In addition to establishing binding to a GPCR in a functionalconformational state, it will also be desirable to determine thefunctional effect of a compound on the GPCR. In particular, the proteinbinding domains or the complexes or the cellular compositions asdescribed herein can be used to screen for compounds that modulate(increase or decrease) the biological activity of the GPCR. The desiredmodulation in biological activity will depend on the GPCR of choice. Thecompounds to be tested can be any small chemical compound, or amacromolecule, such as a protein, a sugar, nucleic acid or lipid.Typically, test compounds will be small chemical compounds, peptides,antibodies or fragments thereof. It will be appreciated that in someinstances high throughput screening of test compounds is preferred andthat the methods as described above may be used as a “library screening”method, a term well known to those skilled in the art. Thus, the testcompound may be a library of test compounds. In particular,high-throughput screening assays for therapeutic compounds such asagonists, antagonists or inverse agonists and/or modulators form parthereof. For high-throughput purposes, compound libraries may be usedsuch as allosteric compound libraries, peptide libraries, antibodylibraries, fragment-based libraries, synthetic compound libraries,natural compound libraries, phage-display libraries and the like.Methodologies for preparing and screening such libraries are known inthe art.

In one preferred embodiment, high-throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic ligands. Such “combinatorial libraries”or “compound libraries” are then screened in one or more assays, asdescribed herein, to identify those library members (particular chemicalspecies or subclasses) that display a desired characteristic activity. A“compound library” is a collection of stored chemicals usually usedultimately in high-throughput screening. A “combinatorial library” is acollection of diverse chemical compounds generated by either chemicalsynthesis or biological synthesis, by combining a number of chemical“building blocks” such as reagents. Preparation and screening ofcombinatorial libraries are well known to those of skill in the art. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics. Thus, in onefurther embodiment, the screening methods as described hereinabovefurther comprise modifying a test compound that has been shown to bindto a GPCR in a functional conformational state, preferably an activeconformational state, and determining whether the modified test compoundbinds to the GPCR when residing in the particular conformation.

In a particular embodiment, the test compound is provided as abiological sample. In particular, the sample can be any suitable sampletaken from an individual. For example, the sample may be a body fluidsample such as blood, serum, plasma, spinal fluid. Alternatively, thesample is tissue or cell extract.

The compounds may bind to the target GPCR resulting in the modulation(activation or inhibition) of the biological function of the GPCR, inparticular, the downstream receptor signaling. This modulation of GPCRsignaling can occur ortho- or allosterically. The compounds may bind tothe target GPCR so as to activate or increase receptor signaling or,alternatively, so as to decrease or inhibit receptor signaling. Thecompounds may also bind to the target GPCR in such a way that they blockoff the constitutive activity of the GPCR. The compounds may also bindto the target complex in such a way that they mediate allostericmodulation (e.g., bind to the GPCR at an allosteric site). In this way,the compounds may modulate the receptor function by binding to differentregions in the GPCR (e.g., at allosteric sites). Reference is made, forexample, to George et al. (2002), Kenakin (2002) and Rios et al. (2001).The compounds hereof may also bind to the target GPCR in such a way thatthey prolong the duration of the GPCR-mediated signaling or that theyenhance receptor signaling by increasing receptor-ligand affinity.Further, the compounds may also bind to the target complex in such a waythat they inhibit or enhance the assembly of GPCR functional homomers orheteromers.

In one embodiment, it is determined whether the compound alters thebinding of the GPCR to a receptor ligand (as defined herein). Binding ofa GPCR to its ligand can be assayed using standard ligand bindingmethods known in the art as described herein. For example, a ligand maybe radiolabeled or fluorescently labeled. The assay may be carried outon whole cells or on membranes obtained from the cells oraqueous-solubilized receptor with a detergent. The compound will becharacterized by its ability to alter the binding of the labeled ligand(see also Example section). The compound may decrease the bindingbetween the GPCR and its ligand, or may increase the binding between theGPCR and its ligand, for example, by a factor of at least two-fold,three-fold, four-fold, five-fold, ten-fold, twenty-fold, thirty-fold,fifty-fold, or one hundred-fold.

Thus, according to more specific embodiments, the complex as used in anyof the above screening methods further comprises a receptor ligand.Preferably, the receptor ligand is chosen from the group comprising asmall molecule, a polypeptide, an antibody or any fragment derivedthereof, a natural product, and the like. More preferably, the receptorligand is a full agonist, or a partial agonist, or an inverse agonist,or an antagonist, as described hereinbefore.

According to a specific embodiment, the protein binding domains hereof,particularly the nanobodies, can also be useful for lead identificationand the design of peptidomimetics. Using a biologically relevant peptideor protein structure as a starting point for lead identificationrepresents one of the most powerful approaches in modern drug discovery.Peptidomimetics are compounds whose essential elements (pharmacophore)mimic a natural peptide or protein in three-dimensional space and thatretain the ability to interact with the biological target and producethe same biological effect. Peptidomimetics are designed to circumventsome of the problems associated with a natural peptide, for example,stability against proteolysis (duration of activity) and poorbioavailability. Certain other properties, such as receptor selectivityor potency, often can be substantially improved. By means of anon-limiting example, the nanobodies hereof may bind with long CDR loopsdeep into the core of the receptor to exert a biological effect. Thesepeptides and their concomitant structures in the nanobody-GPCR complexcan serve as starting points for lead identification and the design ofpeptidomimetics.

Accordingly, the protein binding domains, in particular, the nanobodieshereof, can be useful in screening assays. Screening assays for drugdiscovery can be solid phase or solution phase assays, e.g., a bindingassay, such as radioligand binding assays. In high-throughput assays, itis possible to screen up to several thousand different modulators orligands in a single day in 96-, 384- or 1536-well formats. For example,each well of a microtiter plate can be used to run a separate assayagainst a selected potential modulator, or, if concentration orincubation time effects are to be observed, every five to ten wells cantest a single modulator. Thus, a single standard microtiter plate canassay about 96 modulators. It is possible to assay many plates per day;assay screens for up to about 6,000, 20,000, 50,000, or more, differentcompounds are possible today.

Further, the protein binding domains, such as the nanobodies hereof, canalso be useful in cell-based assays. Cell-based assays are also criticalfor assessing the mechanism of action of new biological targets andbiological activity of chemical compounds. Current cell-based assays forGPCRs include measures of pathway activation (Ca²⁺ release, cAMPgeneration or transcriptional activity); measurements of proteintrafficking by tagging GPCRs and downstream elements with GFP; anddirect measures of interactions between proteins using Fórster resonanceenergy transfer (FRET), bioluminescence resonance energy transfer (BRET)or yeast two-hybrid approaches. Introducing the protein binding domains,in particular, the nanobodies hereof, inside the cell to the relevantcompartment of the cell (intra- or extracellularly) by any means wellknown and commonly used in the art, may lead to new or better cell-basedassays.

In particular, there is a need to “de-orphanize” those GPCRs for which anatural activating ligand has not been identified. The stabilization ofGPCRs in a functional conformational state using the protein bindingdomains hereof enables screening approaches that may be used to identifyligands of “orphan” GPCRs where the natural ligand is unknown. Ligandsof orphan GPCRs may be identified from biological samples such as bloodor tissue extract or from libraries of ligands. For example, variousapproaches to “de-orphanization” have been adopted includingarray-screening against families of known ligands.

The efficacy of the compounds and/or compositions comprising the same,can be tested using any suitable in vitro assay, cell-based assay, invivo assay and/or animal model known per se, or any combination thereof,depending on the specific disease or disorder involved.

Accordingly, in one specific embodiment, a solid support to which isimmobilized a protein binding domain hereof and/or a complex comprisinga protein binding domain and a GPCR in a functional conformationalstate, is provided for use in any of the above screening methods.

In one embodiment, the test compound as used in any of the abovescreening methods is selected from the group comprising a polypeptide, apeptide, a small molecule, a natural product, a peptidomimetic, anucleic acid, a lipid, lipopeptide, a carbohydrate, an antibody or anyfragment derived thereof, such as Fab, Fab′ and F(ab′)2, Fd,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(dsFv) and fragments comprising either a VL or VH domain, a heavy chainantibody (hcAb), a single domain antibody (sdAb), a minibody, thevariable domain derived from camelid heavy chain antibodies (VHH ornanobody), the variable domain of the new antigen receptors derived fromshark antibodies (VNAR), a protein scaffold including an alphabody,protein A, protein G, designed ankyrin-repeat domains (DARPins),fibronectin type III repeats, anticalins, knottins, or engineered CH2domains (nanoantibodies), as defined hereinbefore.

The test compound may optionally be covalently or non-covalently linkedto a detectable label. Suitable detectable labels and techniques forattaching, using and detecting them will be clear to the skilled person,and include, but are not limited to, any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Useful labels include magnetic beads (e.g.,dynabeads), fluorescent dyes (e.g., all Alexa Fluor dyes, fluoresceinisothiocyanate, Texas red, rhodamine, green fluorescent protein and thelike), radiolabels (e.g., ³H, ¹²⁵I, ³S, ¹⁴C, or ³²P), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase), and colorimetric labelssuch as colloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads. Means of detecting such labels arewell known to those of skill in the art. Thus, for example, radiolabelsmay be detected using photographic film or scintillation counters,fluorescent markers may be detected using a photodetector to detectemitted illumination. Enzymatic labels are typically detected byproviding the enzyme with a substrate and detecting the reaction productproduced by the action of the enzyme on the substrate, and colorimetriclabels are detected by simply visualizing the colored label. Othersuitable detectable labels were described earlier within the context ofthe first aspect hereof relating to a protein binding domain.

In a preferred embodiment, the test compound is an antibody or anyfragment derived thereof, as described above, including a nanobody. Forexample, and without the purpose of being limitative, the test compoundmay be an antibody (as defined herein in its broadest sense) that hasbeen raised against a complex comprising a protein binding domain hereofand a GPCR (including variants, as described hereinbefore) in afunctional conformational state, preferably in an active conformationalstate. Methods for raising antibodies in vivo are known in the art.Preferably, immunization of an animal will be done in a similar way asdescribed hereinbefore (immunization with GPCR in presence of receptorligand; see also Example section) with a GPCR in the presence of afunctional conformational state stabilizing protein binding domain, morepreferably, an active state stabilizing protein binding domain. Theinvention also relates to methods for selecting antibodies specific to aGPCR in a functional conformational state, preferably an activeconformational state, involving the screening of expression librariesencoding immunoglobulin genes, or portions thereof, expressed inbacteria, yeast, filamentous phages, ribosomes or ribosomal subunits orother display systems on a complex containing a GPCR and a proteinbinding domain that stabilizes a functional conformational state of theGPCR.

A seventh aspect hereof relates to a kit comprising a protein bindingdomain hereof or a complex hereof or a cellular composition hereof. Thekit may further comprise a combination of reagents such as buffers,molecular tags, vector constructs, reference sample material, as well assuitable solid supports, and the like. Such a kit may be useful for anyof the applications hereof as described herein. For example, the kit maycomprise (a library of) test compounds useful for compound screeningapplications.

Finally, a last aspect hereof is the use of any protein binding domainhereof to isolate amino acid sequences that are responsible for specificbinding to a conformational epitope of a functional conformational stateof a GPCR, in particular, an active conformational state of a GPCR andto construct artificial protein binding domains based on the amino acidsequences. It will be appreciated that in the protein binding domainshereof, the framework regions and the complementarity-determiningregions are known, and the study of derivatives of the protein bindingdomain, binding to the same conformational epitope of a functionalconformational state of a GPCR, in particular, an active conformationalstate of a GPCR, will allow deducing the essential amino acids involvedin binding the conformational epitope. This knowledge can be used toconstruct a minimal protein binding domain and to create derivativesthereof, which can routinely be done by techniques known by the skilledin the art.

The following examples are intended to promote a further understandinghereof. While the present invention is described herein with referenceto illustrated embodiments, it should be understood that the inventionis not limited hereto. Those having ordinary skill in the art and accessto the teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the claims attached herein.

EXAMPLES Protein Binding Domains Stabilizing Functional ConformationalStates of Human β₂AR Example 1. Immunization, Library Construction andInitial Screening

To obtain in vivo matured nanobodies against β₂AR, a llama (Llama glama)was immunized with recombinant β₂AR truncated at Gly365 (β₂AR-365) toexclude an immune response to the carboxyl terminus. β₂AR-365 wasexpressed in insect cells and antigen was reconstituted as previouslydescribed (Day et al. 2007). After six weekly administrations of thereconstituted truncated agonist-bound receptor, lymphocytes wereisolated from the blood of the immunized llama and a phage libraryprepared and screened as described in Materials and Methods to theExamples (see further). Two screens identified conformational nanobodiesthat recognize the native β₂AR, but not the denaturated receptor.

Example 2. Selection of Conformational-Specific Nanobodies by ELISA

In a first screen we compared the binding of the nanobodies on thenative and heat denatured β₂AR antigen in an ELISA. For each nanobody,one well was coated with phospholipid vesicles containing agonist-boundβ₂AR-365 (0.1 μg protein/well). Next, this plate was incubated at 80° C.for two hours. Next, another well of the same plate was coated withphospholipid vesicles containing agonist-bound β₂AR-365 (0.1 μgprotein/well) without heating. All of the nanobodies were able toselectively bind the native receptor but not the heat inactivatedreceptor, indicating that 16 binders recognize conformational epitopes.

Example 3. Selection of Conformational-Specific Nanobodies by Dot Blot

In a next screen we compared the specificity of the nanobodies for anative agonist-bound β₂AR receptor, versus a native inverseagonist-bound receptor, versus an SDS denaturated receptor by dot blotanalysis. The screen identified 16 different conformational nanobodiesthat recognize native agonist-bound β₂AR-365, but not the inverseagonist, or the heat denatured receptor (FIG. 2 (dot blots)).

Example 4. Selection of Nanobodies with G Protein-Like Behavior

The initial screen identified 16 clones that recognized nativeagonist-bound β₂AR, but not heat denatured receptor. Our next goal wasto identify nanobodies that had G protein-like behavior. The β₂ARpreferentially couples to Gs and agonist binding enhances G proteininteractions. Moreover, in the presence of Gs, the β₂AR binds agonistwith higher affinity. Therefore, we looked at (1) the effect of agoniston nanobody binding to the β₂AR using size exclusion chromatography, (2)the effect of nanobodies on β₂AR agonist binding affinity in membranesand (3) the effect of nanobodies on β₂AR conformational changes asmonitored by the environmentally sensitive monobromobimane (mBBr)fluorophore.

Example 5. Nanobodies with G Protein-Like Behavior Specifically Bind toPurified Agonist-Bound Receptor

Purified nanobodies were incubated with purified, detergent-solubilizedβ₂AR receptor in the presence of an agonist or an inverse agonist thatstabilizes an inactive conformation. The mixture was then analyzed bySize Exclusion Chromatography (SEC), which separates protein on thebasis of size. Seven of the nanobodies bound to purified β₂AR andmigrated as a complex on SEC only in the presence of agonist and not aninverse agonist (an example is shown in FIG. 1, Panel a, FIGS. 3A, 3B,and FIG. 4). The remaining nanobodies did not bind with sufficient highaffinity to shift the mobility of the β₂AR on SEC.

Example 6. Nanobodies with G Protein-Like Behavior Enhance the Affinityof β₂AR for Agonists

Many GPCRs exhibit higher agonist binding affinity when complexed with Gprotein. This is attributed to the cooperative interaction betweenagonist occupied receptor and G protein. Our SEC experiments provideevidence that seven nanobodies preferentially bind to agonist occupiedβ₂AR and may therefore stabilize an active state in a manner similar tothe G protein Gs. Agonist competition binding experiments were performedin the presence and absence of these seven nanobodies. The affinity ofthe β₂AR for the agonist (isoproterenol) was enhanced two- tothirty-fold in the presence of nanobodies 65, 67, 69, 71, 72, 80 and 84(FIG. 1, Panel b, and Table 1). In contrast, Nb80 does not increase theaffinity of β₂AR or β₂AR-T4L for the inverse agonist ICI-118,551 (ICI)(FIG. 15).

Example 7. Nb80 and the G Protein Induce Similar Conformational Changesat the Cytoplasmic Domain of TM6

The recent crystal structure of opsin as well as biophysical studies onrhodopsin (Park et al. 2008) and the β₂AR (Yao et al. 2009) show thatthe cytoplasmic end of transmembrane segment 6 (TM6) undergoesconformational changes upon agonist binding that are required for Gprotein coupling. To investigate the effect of nanobodies on movement ofthe cytoplasmic domain of TM6 we labeled purified β₂AR at C265 withmonobromobimane (mBB-β₂AR). We previously showed that both agonistbinding and G protein coupling-induced changes in the fluorescence ofmBB-β₂AR compatible with an outward movement of TM6 (Yao et al. 2008) asobserved in the opsin crystal structure (Park et al. 2008). In mBB-β₂AR,the addition of agonist together with G protein results in largerfluorescent changes than either agonist or G protein alone. This iscompatible with the cooperative interactions observed in agonistcompetition binding assays (Yao et al. 2009). Similarly, relativelysmall changes in fluorescence were observed in mBB-β₂AR with theaddition of either the agonist isoproterenol alone or Nb80 alone;however, larger changes were observed when both agonist and nanobodywere added together (FIG. 1, Panel c). Comparable results were observedfor nanobodies 65, 67, 69, 71, 72 and 84 (FIGS. 5A-5F).

Example 8. Characterization of the Nanobody-Stabilized β₂AR Active State

We then compared the effect of Nb80 with Gs on β₂AR structure andagonist binding affinity. β₂AR was labeled at the cytoplasmic end of TM6at C265 with monobromobimane and reconstituted into HDL particles. TM6moves relative to TM3 and TM5 upon agonist activation (FIG. 6, Panel A),and we have previously shown that the environment around bimanecovalently linked to C265 changes with both agonist binding and Gprotein coupling, resulting in a decrease in bimane intensity and a redshift in λ_(max) (Yao et al. 2009). The change in bimane fluorescence iscompatible with movements of TM6 similar to those observed in rhodopsinby DEER spectroscopy and in the structure of low pH opsin. As shown inFIG. 6, Panel B, the catecholamine agonist isoproterenol and Gs bothstabilize an active-like conformation, but the effect of Gs is greaterin the presence of isoproterenol, consistent with the cooperativeinteractions of agonist and Gs on β₂AR structure. Nb80 alone has aneffect on bimane fluorescence and λ_(max) of unliganded β₂AR this issimilar to that of Gs (FIG. 6, Panel C). This effect was not observed inβ₂AR bound to the inverse agonist ICI-118,551. The effect of Nb80 wasincreased in the presence of 10 μM isoproterenol. These results showthat Nb80 does not recognize the inactive conformation of the β₂AR, butbinds efficiently to agonist occupied β₂AR and produces a change inbimane fluorescence that is indistinguishable from that observed in thepresence of Gs and isoproterenol.

FIG. 6, Panels D and E, show the effect of Gs and Nb80 on agonistaffinity for β₂AR. β₂AR was reconstituted into HDL particles and agonistcompetition binding experiments were performed in the absence orpresence of Nb80 and Gs. In the absence of either protein, isoproterenolhas an inhibition constant (Ki) of 107 nM. In the presence of Gs twoaffinity states are observed, because not all of the β₂AR is coupled toGs. In the Gs-coupled state, the affinity of isoproterenol increases by100-fold (Ki=1.07 nM) (FIG. 6, Panel D, and Table 4). Similarly, in thepresence of Nb80 the affinity of isoproterenol increases by 95-fold(Ki=1.13 nM) (FIG. 6, Panel E, and Table 4). These binding data suggestthat Nb80 stabilizes a conformation in WT β₂AR that is very similar tothat stabilized by Gs, such that the energetic coupling of agonist andGs binding is faithfully mimicked by Nb80.

The high-resolution structure of the inactive state of the β₂AR wasobtained with a β₂AR-T4L fusion protein. We previously showed thatβ₂AR-T4L has a higher affinity for isoproterenol than WT β₂AR (Rosenbaumet al. 2007). Nevertheless, in the presence of Nb80 the affinityincreased by 60-fold, resulting in an affinity (Ki=0.56 nM) comparableto that of WT β₂AR bound to Nb80 (FIG. 6, Panel F, and Table 4). Whilewe cannot study G protein coupling in β₂AR-T4L due to steric hindranceby T4L, the results show that T4L does not prevent binding of Nb80, andthe nearly identical Ki values for agonist binding to wild-type β₂AR andβ₂AR-T4L in the presence of Nb80 suggest that Nb80 stabilizes a similarconformation in these two proteins.

Example 9. Nanobodies Facilitate Crystallization of Agonist-Bound β₂AR

The β₂AR was originally crystallized bound to the inverse agonistcarazolol using two different approaches. The first crystals wereobtained from β₂AR bound to a Fab fragment that recognized an epitopecomposed of the amino and carboxyl terminal ends of the thirdintracellular loop connecting TMs 5 and 6 (Rasmussen et al. 2007). Inthe second approach, the third intracellular loop was replaced by T4lysozyme (β₂AR-T4L) (Rosenbaum et al. 2007). Efforts to crystallizeβ₂AR-Fab complex and β₂AR-T4L bound to different agonists failed toproduce crystals of sufficient quality for structure determination.

We, therefore, attempted to crystallize agonist-bound β₂AR and β₂AR-T4Lin complex with Nb80. While crystals of both complexes were obtained inlipid bicelles and lipidic cubic phase (LCP), high-resolutiondiffraction was obtained from crystals of β₂AR-T4L-Nb80 grown in LCP.These crystals grew at pH 8.0 in 39% to 44% PEG400, 100 mM Tris, 4%DMSO, and 1% 1,2,3-heptanetriol.

Example 10. Nb80 Contributes to the Packing of β₂AR in a Crystal Lattice

High-resolution diffraction was obtained from crystals of β₂AR-T4L-Nb80grown in LCP. These crystals grew at pH 8.0 in 39% to 44% PEG400, 100 mMTris, 4% DMSO, and 1% 1,2,3-heptanetriol.

A merged data set at 3.5 Å was obtained from 23 crystals (Table 5). Thestructure was solved by molecular replacement using the structure of thecarazolol-bound β₂AR and a nanobody as search models. FIG. 7 shows thepacking of the β₂AR-T4L-Nb80 complex in the crystal lattice. Nb80 bindsto the cytoplasmic end of the β₂AR, with the thirdcomplementarity-determining region (CDR) loop projecting into the coreof the receptor. The β₂AR-nanobody complexes are arranged with the lipidbilayers approximately parallel to the be plane of the crystal. Two-foldsymmetry-related nanobody molecules interact along the a axis togenerate a tightly packed lattice in this direction. Within the bilayer,receptor molecules interact in an antiparallel arrangement with TM1,2,3and 4 of one β₂AR molecule packing against TM4 and 5 of the adjacentmolecule. Contacts are also made between helix 8 and TM5 of parallellattice neighbor along the b axis, and between the extracellular portionof TM1 and the cytoplasmic end of TM6 of a third, antiparallel neighbor.The packing is weakest along the c axis, which may be due in part tonon-specific interactions of the T4L with neighboring receptor and/ornanobody molecules. There is no interpretable electron density for theT4L, but given the visible ends of TM5 and TM6 the position of T4L ishighly constrained. Presumably T4L adopts a number of orientationsrelative to the receptor, and perhaps a range of internal conformationsdue to its hinge motion (Zhang et al. 1995), that average out itsdensity. Nonetheless, T4L likely contributes to the structure of thecrystal since we were unable to produce crystals of the nativeβ₂AR-nanobody complex under these conditions, although it is possiblethat the flexible loop that connects TM5 and TM6 in the native receptorprevents lattice formation.

Example 11. Structure of a Nanobody-Stabilized Active State of β₂AR

FIG. 8 compares the inactive β₂AR structure (from the carazolol-boundβ₂AR-T4L structure) with the active state of β₂AR. The largestdifferences are found at the cytoplasmic face of the receptor, withoutward displacement of TM5 and TM6 and an inward movement of TM7 andTM3 in the β₂AR-T4L-Nb80 complex relative to the inactive structure(FIG. 8, Panels A and B). There are relatively small changes in theextracellular surface (FIG. 8, Panel C). The second intracellular loop(ICL2) between TM3 and TM4 adopts a two-turn alpha helix, similar tothat observed in the turkey β₁AR structure (Warne et al. 2008). Theabsence of this helix in the inactive β₂AR structure may reflect crystallattice contacts involving ICL2.

FIG. 9, Panels A-C, show in greater detail the interaction of Nb80 withthe cytoplasmic side of the β₂AR. An eight amino acid sequence of CDR 3penetrates into a hydrophobic pocket formed by amino acids from TMsegments 3, 5, 6 and 7. A four amino acid sequence of CDR1 providesadditional stabilizing interactions with cytoplasmic ends of TM segments5 and 6. FIG. 9, Panel D, compares the cytoplasmic surface of active andinactive conformations of the β₂AR. CDR3 occupies a position similar tothe carboxyl terminal peptide of transducin in opsin (Scheerer et al.2008) (FIG. 10). The majority of interactions between Nb80 and the β₂ARare mediated by hydrophobic contacts.

When comparing the active and inactive structures, the largest change isobserved in TM6, with an 11.4 Å movement of the helix at Glu268^(6,30)(part of the ionic lock) (superscripts in this form indicateBallesteros-Weinstein numbering for conserved GPCR residues (Ballesterosand Weinstein 1995) (FIG. 9, Panel D). This large change is effected bya small clockwise rotation of TM6 in the turn preceding the conservedPro288^(6,50), enabled by the interrupted backbone hydrogen bonding atthe proline and repacking of Phe282⁶⁻⁴⁴ (see below), which swings thehelix outward.

The changes in the active β₂AR-T4L-Nb80 relative to the inactivecarazolol-bound β₂AR-T4L are remarkably similar to those observedbetween rhodopsin and opsin (Scheerer et al. 2008; Park et al. 2008)(FIG. 9, Panel E, and FIG. 10). The salt bridge in the ionic lockbetween highly conserved Arg131^(3,50) and Asp/Glu130^(3,49) is broken.In opsin, Arg135^(3,50) interacts with Tyr223^(5,58) in TM5 and abackbone carbonyl of the transducin peptide. Arg131^(3,50) of β₂ARlikewise interacts with a backbone carbonyl of CDR3 of Nb80. However,Nb80 precludes an interaction between Arg131^(3,50) and Tyr219^(5,58),even though the tyrosine occupies a similar position in opsin and theactive conformation of NAR-T4L-Nb80. As in opsin, Tyr326^(7,53) of thehighly conserved NPxxY sequence moves into the space occupied by TM6 inthe inactive state. In inactive carazolol-bound β₂AR-T4L we observed anetwork of hydrogen bonding interactions involving highly conservedamino acids in TMs 1, 2, 6 and 7 and several water molecules (Rosenbaumet al. 2007). While the resolution of the β₂AR-T4L-Nb80 is inadequate todetect waters, it is clear that the structural changes we observe wouldsubstantially alter this network.

In contrast to the relatively large changes observed in the cytoplasmicdomains of β₂AR-T4L-Nb80, the changes in the agonist-binding pocket arefairly subtle. Trp^(6,48) is highly conserved in Family A GPCRs, and ithas been proposed that its rotameric state plays a role in GPCRactivation (rotamer toggle switch) (Shi et al. 2002). We observe nochange in the side chain rotamer of Trp286^(6,48) in TM6, which liesnear the base of the ligand-binding pocket, although its position shiftsslightly due to rearrangements of nearby residues Ile121^(3,40) andPhe282^(6,44). While there is spectroscopic evidence for changes in theenvironment of Trp^(6,48) upon activation of rhodopsin (Ahuja et al.2009), a rotamer change is not observed in the crystal structures ofrhodopsin and low-pH opsin. Moreover, recent mutagenesis experiments onthe histamine receptor demonstrate that Trp^(6,48) is not required foractivation of the 5HT4 receptor by serotonin (Pellissier et al. 2009).

It is interesting to speculate how the small changes around theagonist-binding pocket are coupled to much larger structural changes inthe cytoplasmic regions of TMs 5, 6 and 7 that facilitate binding ofNb80 and Gs. A potential conformational link is shown in FIG. 11.Agonist interactions may stabilize an active receptor conformation thatincludes a 2.1 Å inward movement of TM5 at position 207^(5,46) and 1.4 Åinward movement of the conserved Pro211^(5,50) relative to the inactivestructure. In the inactive state, the relative positions of TM5, TM3, TM6 and TM7 are stabilized by interactions between Pro211^(5,50),Ile121^(3,40), Phe282^(6,44) and Asn318^(7,45). The position ofPro211^(5,50) observed in the active state is incompatible with thisnetwork of interactions, and Ile121^(3,40) and Phe282^(6,44) arerepositioned, with a rotation of TM6 around Phe282^(6,44) leading to anoutward movement of the cytoplasmic end of TM6.

Although some of the structural changes observed in the cytoplasmicdomains of the β₂AR-T4L-Nb80 complex arise from specific interactionswith Nb80, the fact that Nb80 and Gs induce or stabilize similarstructural changes in the β₂AR, as determined by fluorescencespectroscopy and by agonist binding affinity, suggests that Nb80 and Gsrecognize similar agonist stabilized conformations. The observation thatthe cytoplasmic domains of rhodopsin and the β₂AR undergo similarstructural changes upon activation provides further support that theagonist-bound β₂AR-T4L-Nb80 represents an active conformation and isconsistent with a conserved mechanism of G protein activation.

However, the mechanism by which agonists induce or stabilize theseconformational changes likely differs for different ligands and fordifferent GPCRs. The conformational equilibria of rhodopsin and β₂ARdiffer, as shown by the fact that rhodopsin can adopt a fully activeconformation in the absence of a G protein whereas β₂AR cannot. Thus,the energetics of activation and conformational sampling can differamong different GPCRs, which likely gives rise to the variety of ligandefficacies displayed by these receptors. An agonist need only disruptone key intramolecular interaction needed to stabilize the inactivestate, as constitutive receptor activity can result from singlemutations of amino acids from different regions of GPCRs (Parnot et al.2002). Thus, disruption of these stabilizing interactions either byagonists or mutations lowers the energy barrier separating inactive andactive states and increases the probability that a receptor can interactwith a G protein.

In conclusion, these above results demonstrate the ability to generatenanobodies that recognize and stabilize an agonist-bound state of aGPCR. In the case of the β₂AR, this nanobody-stabilized state isfunctionally similar to the state stabilized by the G protein Gs.Finally, nanobodies facilitated the formation of diffraction qualitycrystals. This approach is now applied to other GPCRs and other membraneproteins.

Example 12. Nb80 Stabilizes the Active State Conformation of Members ofthe Adrenergic Receptor Family

Active state stabilizing Nanobodies that are cross-reactive to relatedreceptors can be used as a tool to stabilize a conformational state ofthose related receptors. To demonstrate this principle, we analyzed ifNb80 selectively binds the active conformation of the human β₁ARreceptor. β₁AR and β₂AR are closely related adrenergic receptors. Usingthe PISA server (Krissinel & Henrick, 2007) and based on the crystalstructure of the Nb80-02AR complex, 30 β₂AR AA residues were identifiedto interact with Nb80 in the β₂AR-Nb80 interface. An amino acid sequencealignment (FIG. 17) indicates that 28 out of these 30 residues involvedin the Nb80 interaction are conserved among β₁AR and β₂AR. The remaininginterface residues are lysines in β₂AR and have been substituted byarginines in β₁AR corresponding to conserved substitutions (shown ingrey boxes in FIG. 17). It thus appears that both receptors share a verysimilar binding site for Nb80. Based on this analysis, we also measuredthe effect of Nb80 on the affinity of β₁AR for the agonist isoproterenoland the inverse agonist CGP20712A (FIG. 18). As for β₂AR, Nb80 alsoinduced an increased affinity of isoproterenol for β₁AR (FIGS. 18A and18C). Nb80 does not change the affinity for the antagonist CGP20712A(FIG. 18D), demonstrating the selective stabilization of the activestate conformation of β₁AR by Nb80. No such effect of Nb80 was observedon unrelated receptors like the dopamine D1 receptor (data not shown).

Example 13. β₂AR Active State Stabilizing Nanobodies are Excellent Toolsfor Improved Agonist Screening

Many GPCRs exhibit higher agonist binding affinity when complexed with Gprotein. This is attributed to the cooperative interaction betweenagonist occupied receptor and G protein. Nanobodies with G protein-likebehavior likely enhance the affinity of β₂AR for agonists (see Example6). This behavior may have important implications in the discovery ofnew agonists. For example, Nbs with G protein-like behavior may increasethe apparent affinity of GPCRs for agonists, compared to antagonists.This may cause a bias towards agonists among the hits when a compoundlibrary is screened against such GPCR-Nb complex. To evaluate theapplicability of this approach, we analyzed the effect of the β₂ARactive state stabilizing Nanobody Nb80 on the interaction of β₂AR withseveral well known β₂AR ligands in competition radioligand bindingexperiments (see materials and methods). Ten agonists and fiveantagonists were tested: (-)isoproterenol HCl (agonist), (-)-alprenolol(antagonist), salbutamol (partial agonist), ICI118551 (inverse agonist),carvedilol (antagonist), CGP12177A (antagonist), salmeterol Xinafoate(full agonist), terbutaline hemisulfate salt (partial agonist),dobutamine hydrochloride (partial agonist), metaproterenol hemisulfatesalt (agonist), procaterol hydrochloride (agonist), ritodrinehydrochloride (agonist), fenoterol hydrobromide (full agonist),formoterol fumarate dihydrate (agonist) and timolol maleate salt(antagonist), all purchased via Sigma Aldrich.

Ligand competition binding experiments were performed in the presenceand absence of 500 nM of Nb80 on commercial membranes containingfull-length human β₂AR. ³H-dihydroalprenolol (DHA) was used as thecompeting radioligand. Representative examples of ligand competitionbinding experiments are shown in FIGS. 16A and 16B. For all compounds,IC50 values were obtained in the presence of excess Nb80 and compared tothe IC50s obtained in the presence of an irrelevant nanobody (negativecontrol). (Table 6). Consistent with Examples 6, 8 and 12, we observe anincrease in potency for all agonists when β₂AR is in complex with Nb80.Such effect is not observed for antagonists or inverse agonists.

Example 14. Compound Library Screening Using a β₂AR Active StateStabilizing Nanobody

Locking the GPCR in a particular conformation has an advantage overusing a non-conformationally stabilized target (representing arepertoire of conformations) in screening assay with the aim to identifycompounds against that particular conformation, the so-called“druggable” target conformation. The conformational selective Nb80allows the use of wild-type receptors consequently minimizing thepotential risk of artificial conformational changes as a result ofsite-directed mutagenesis.

In order to demonstrate whether Nb80 facilitates the identification ofligands that selectively bind β₂AR in its active conformation, afragment library consisting of approximately 1500 distinct low molecularweight compounds (<300 Da) is screened for agonists using a competitionradioligand binding assay. For this assay, membranes containing fulllength β₂AR are preincubated with Nb80 or an irrelevant Nb (negativecontrol) and added to 96-well plates containing library compounds and 2nM of ³H-dihydroalprenolol (DHA) radioligand (see materials andmethods). Library compounds that significantly displace the radioligandin the sample containing β₂AR in complex with Nb80 as compared to thesample containing the irrelevant nanobody are compounds thatpreferentially bind the active conformation of the receptor. Librarycompounds that selectively bind the active conformation of the receptorhave a high propensity to behave as agonists because orthosteric orallosteric stabilization of the active conformation of the GPCR elicitsbiological responses. Selected library compounds have to be furtherscreened for agonism by measuring for example by G protein coupling,downstream signaling events ore physiological output.

Example 15. Thermostabilization of the β₂AR Receptor with Nanobodies

Structural and functional studies on integral membrane proteins havelong been hampered by their instability in detergent. Althoughexpression and purification methods are appearing that allow for thegeneration of milligram quantities, achieving stability with thesemolecules is perhaps the most difficult hurdle to overcome. Purificationnecessitates a release of the membrane protein from the lipid bilayer bydetergent solubilization, a process during which hydrophobic surfaces ofthe protein are coated with surfactant monomers to form aprotein-detergent complex. However, the detergent belt formed around theprotein is a poor replacement for the lipid bilayer. Thus,solubilization of membrane proteins often results in destabilization,unfolding and subsequent aggregation. Thermostabilization of membraneproteins can be achieved through site directed mutagenesis (Zhou &Bowie, 2000; Magnini et al. 2008). Here, we show that binding ofconformational selective nanobodies represents an innovative alternativefor the thermostabilization of detergent-solubilized GPCRs.

The effect of conformational selective nanobodies on the thermostabilityand the subsequent aggregation of the β₂AR receptor was analyzed using afluorescent thermal stability assay (FIG. 13) and size exclusionchromatography (FIGS. 14A and 14B). For these experiments, recombinantβ₂AR was expressed in 519 insect cells, solubilized 1% dodecylmaltoside,100 mM NaCl, 20 mM Hepes pH 7.5 and protease inhibitors, and purified byM1 FLAG affinity chromatography (see Material and Methods to theExamples).

The fluorescent thermal stability assay makes use of the thiol-specificfluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide(CPM) to measure the chemical reactivity of the native cysteinesembedded in the protein interior as a sensor for the overall integrityof the folder state of membrane proteins (Alexandrov et al. 2008). Forthe fluorescent thermal stability assay, detergent-solubilized receptor(in 0.1% dodecylmaltoside, 100 mM NaCl and 20 mM Hepes pH 7.5) waspre-incubated with isoproterenol (10 μM) in the presence or absence of a2:1 molar excess of Nb80 for one hour at RT. Samples were then mixedwith CPM fluorophore and incubated for two minutes at temperaturesranging from 10° C. to 94° C. and fluorescence emission was collected.Experiments were performed in triplicate and resulted in melting curvesresembling the stability profiles obtained for the GPCR APJ (Alexandrovet al. 2008). Unfolding transitions could be described by a simpletwo-state model, including the native (folded) and the denaturatedstate, representing the lower and upper plateaus of the melting curves.Comparison of the melting curve of the agonist-bound receptor with themelting curve of agonist-bound receptor in complex with Nb80 indicatesthat the nanobody stabilizes the active conformation of β₂AR byincreasing the melting temperature (Tm) of the agonist-bound receptor by12° C.

We also analyzed the effect of Nb80 on the thermal unfolding andaggregation of the agonist-bound receptor by size exclusionchromatography (SEC). For this experiment, dodecylmaltoside-solubilizedreceptor was pre-incubated with isoproterenol (10 μM) in the presence orabsence of a 2:1 molar excess of Nb80 for 45 minutes at RT. Samples werenext incubated for 10 minutes at increasing temperatures andsubsequently analyzed by SEC (FIGS. 14A and 14B). Comparison of thedifferent chromatograms indicates that Nb80 protects the agonist-boundreceptor against temperature-induced aggregation.

Protein Binding Domains Stabilizing Functional Conformational States ofRat Angiotensin II Type 1a Receptor (AT1aR) Example 16. Immunization,Library Construction and Initial Screening

To obtain in vivo matured nanobodies against rat AT1aR, two llamas(Llama glama) were immunized with a recombinant AT1aR-T4lysozyme (T4L)fusion truncated after Lys318 to exclude an immune response to thecarboxyl terminus. AT1aR was expressed in insect cells (Shluka et al.2006) and antigen was reconstituted in lipid vesicles as previouslydescribed (Day et al. 2007). One llama was immunized with angiotensin-(unbiased agonist-) bound receptor, one llama was immunized with theβ-arrestin biased ligand TRV023 (Violin et al. 2010) bound to thereceptor. After six weekly administrations of the reconstitutedtruncated agonist-bound receptor, lymphocytes were isolated from theblood of the immunized llama and a phage library prepared and screenedas described in Materials and Methods to the Examples. Solid-phaseELISAs identified nanobodies that recognize the AT1a receptor.

Example 17. Selection of Conformational-Specific Nanobodies by ELISA

In a first screen we compare the binding of the purified nanobodies onthe native and heat denatured rat AT1aR antigen in an ELISA. For eachnanobody, one well is coated with receptor. Next, this plate isincubated at 80° C. for two hours. Next, another empty well of the sameplate is coated with receptor without heating. Nanobodies that are ableto selectively bind the native receptor but not the heat inactivatedreceptor recognize conformational epitopes.

Example 18. Selection of Conformational State-Specific Nanobodies by DotBlot

In a next screen we compare the binding of those nanobodies that bindconformational epitopes to an agonist-bound AT1aR receptor versus anantagonist-bound receptor by dot blot analysis. This screen identifiesnanobodies that selectively recognize a conformation of an agonist-bound(active state) versus antagonist-bound (inactive state) receptor.

Example 19. Screening for AT1aR Nanobodies Selectively Stabilizing anActive Conformation of the Receptor

In addition to the binding assay for AT1aR specificity (ELISA), purifiedNanobodies are evaluated in a radioligand competition experiment similarto the radioligand assays described in Examples 6, 12, and 13.Nanobodies that increase the affinity of AT1aR to an agonist areconsidered to stabilize an active conformation of the receptor.

Protein Binding Domains Stabilizing Functional Conformational States ofRat M3-Muscarinic Receptor Example 20. Immunization, LibraryConstruction and Initial Screening

To obtain in vivo matured nanobodies against rat M3R, a llama (Llamaglama) was immunized with a recombinant M3R-T4lysozyme (M3R-T4L) fusiontruncated at both N- and C-terminal sites to exclude an immune responseto the termini. In M3R-T4L the third intracellular loop was replaced byT4lysosyme. M3R-T4L was expressed in insect cells and antigen wasreconstituted as previously described (Day et al. 2007). After sixweekly administrations of the reconstituted antagonist- (tiotropium-)bound receptor, lymphocytes were isolated from the blood of theimmunized llama and a phage library prepared and screened as describedin Materials and Methods to the Examples.

Example 21. Selection of Conformational-Specific Nanobodies by ELISA

In a first screen we compare the binding of the purified nanobodies onthe native and heat denatured rat M3R antigen in an ELISA. For eachnanobody, one well is coated with receptor. Next, this plate isincubated at 80° C. for two hours. Next, another empty well of the sameplate is coated with receptor without heating. Nanobodies that are ableto selectively bind the native receptor but not the heat inactivatedreceptor recognize conformational epitopes.

Example 22. Selection of Conformational State-Specific Nanobodies by DotBlot

In a next screen we compare the binding of those nanobodies that bindconformational epitopes to an agonist-bound M3 receptor versus anantagonist-bound receptor by dot blot analysis. This screen identifiesnanobodies that selectively recognize a conformation of an agonist-bound(active state) versus antagonist-bound (inactive state) receptor.

Example 23. Screening for M3R Nanobodies Selectively Stabilizing anActive Conformation of the Receptor

In addition to the binding assay for M3R specificity (ELISA), purifiedNanobodies are evaluated in a radioligand competition experiment similarto the radioligand assays described in Examples 6, 12, and 13.Nanobodies that increase the affinity of M3R to an agonist areconsidered to stabilize an active conformation of the receptor.

Materials and Methods to Examples β₂AR Preparation

β₂AR truncated after amino acid 365 (β₂AR-365) having an amino terminalFlag epitope tag was expressed in Sf9 insects cells and purified bysequential M1 antibody and alprenolol affinity chromatography aspreviously described (Kobilka 1995). Purified β₂AR-365 was immobilizedon a Flag column (Sigma) and equilibrated with 10 column volumes of amixture of 5 mg/ml DOPC (Avanti Polar Lipids) and 0.5 mg/ml Lipid A(Sigma) in 1% (w/v) octylglucoside (Anatrace), 100 mM NaCl, 20 mM HepespH 7.5, 2 mM CaCl2 and 1 μM agonist (e.g., isoproterenol). The β₂AR wasthen eluted in the same buffer containing EDTA. The concentration of theeluted β₂AR was adjusted to 5 mg/ml. This usually involved diluting theprotein with the same buffer, but occasionally required concentratingthe protein up to two-fold with an Amicon ultrafiltration cell (100 kDapore). The protein was then dialyzed against phosphate buffered salinecontaining 1 μM agonist at 4° C. to remove detergent. The reconstitutedprotein was stored at −80° C. prior to use for immunization.

AT1aR Preparation

AT1aR with T4lysozyme fusion in the third loop was truncated after aminoacid 318 (AT1aR-318). This construct has an amino terminal Flag epitopetag and a C-terminal tag of ten histidines. AT1aR-318 was expressed inTni insect cells and solubilized in 20 mM Hepes, pH7.4, 1 M NaCl and0.5% MNG for two hours at room temperature. The receptor was purified bysequential Ni-NTA and FLAG-M1 antibody affinity chromatography. PurifiedAT1aR was reconstituted in a mixture of 5 mg/ml DOPC (Avanti PolarLipids) and 0.5 mg/ml Lipid A (Sigma) in 1% (w/v) octylglucoside(Anatrace), 100 mM NaCl, 20 mM Hepes pH 7.5, and 100 μM agonist (e.g.,angiotensin II). The concentration of the eluted At1aR was adjusted to1-2 mg/ml. The protein was then dialyzed against phosphate bufferedsaline containing 100 agonist at 4° C. to remove detergent. Thereconstituted protein was stored at −80° C. prior to use forimmunization.

M3 Receptor Preparation

M₃-muscarinic receptor with an amino terminal FLAG epitope tag andcarboxy-terminal hexahistidine tag was expressed in Sf9 insect cells inthe presence of 1 μM atropine (Vasudevan et al. 1995). Receptor eitherhad intracellular loop 3 deleted (M3RΔi3) or substituted with T4lysozyme (M3R-T4L). Cells were centrifuged and then lysed by osmoticshock, and protein was solubilized in 1% dodecylmaltoside, 0.1%cholesterol hemisuccinate, 750 mM sodium chloride, 20 mM HEPES pH 7.5.Solubilized receptor was then purified by nickel affinity chromatographyfollowed by FLAG affinity chromatography. Purified protein was thenseparated by size exclusion chromatography to select monomeric receptor,which was reconstituted as described (Day et al. 2007).

Nanobody Selection Against β₂AR

A single llama received six weekly administrations of the reconstitutedtruncated β₂AR. Lymphocytes were isolated from the blood of theimmunized llama and total RNA was prepared from these cells. The codingsequences of the nanobody repertoire were amplified by an RT-PCR andcloned into the phage display vector pMES4 (genbank GQ907248) (Conrathet al. 2001). β₂AR-specific phages were enriched by in vitro selectionon Maxisorp (Nunc) 96-well plates coated with the reconstituted β₂AR-365receptor. Antigen-bound phages were recovered from antigen-coated wellseither with thriethylamine pH11 and neutralized with Tris-HCl pH7 or bythe addition of freshly grown TG1 E. coli cells. After two rounds ofbio-panning, 96 individual colonies were randomly picked and thenanobodies produced as a soluble HIS-tagged protein in the periplasm ofthe TG1 cells. Solid-phase ELISAs identified 16 different conformationalnanobodies that recognize native agonist-bound β₂AR-365, but not theheat denatured receptor.

Nanobody Selection Against AT1aR

One single llama received six weekly administrations of thereconstituted AT1aR-318 bound to its agonist angiotensin. Another singlellama received six weekly administrations of AT1aR-318 bound to a biasedagonist TRV023. After immunization, lymphocytes from each llama wereisolated separately from the blood. Total RNA was prepared from thesecells. From each sample the coding sequences of the nanobody repertoirewere amplified by RT-PCR and cloned separately (Conrath et al. 2001)into the phage display vector pMESy4-vector to generate two independentlibraries. pMESy4 is a derivative of pMES4 (genbank GQ907248) carrying aC-terminal His6tag followed by the amino acids EPEA (De Genst et al.2010, J Mol. Biol. 402:326-343).

AT1aR-specific phage was enriched by in vitro selection on Maxisorp(Nunc) 96-well plates coated with the reconstituted truncated AT1aRreceptor (a variant of the recombinant receptor without the T4Linsertion) bound to angiotensin or to TRV023, respectively.Antigen-bound phage was recovered from antigen-coated wells by trypsindigestion. After two rounds of bio-panning, 92 individual colonies (46on AT and 46 on AT1aR-TRV023) were randomly picked and the nanobodieswere produced as a soluble HisIS-EPEA-tagged protein in the periplasm ofthe TG1 cells.

Nanobody Selection Against M3R

One single llama received six weekly administrations of reconstitutedtruncated M3R-T4L bound to the antagonist tiotropium. Afterimmunization, lymphocytes from this llama were isolated from the bloodand total RNA was prepared from these cells. The coding sequences of thenanobody repertoire were amplified by RT-PCR and cloned (Conrath et al.2001) into the phage display vector pMESy4-vector.

To enrich for M3R-specific phages different in vitro selectionstrategies were followed using different formats of the antigen in thepresence of the agonist carbachol or the antagonist quinuclidinylbenzylate (QNB). Antigen formats include virus-like particles (VLPs)carrying the rat M3RΔi3 receptor (i.e., the M3 receptor with a deletionin the third intracellular loop), membranes of human CHO cellscontaining M3R (Perkin Elmer), recombinant reconstituted M3R-T4L, orrecombinant reconstituted M3RΔi3. Optionally, VLPs carrying the ratM3RΔi3 receptor or the human M3R membranes were captured by wheat germagglutinin coated on Maxisorp (Nunc) 96-well plates. Antigen-bound phagewas recovered from antigen-coated wells by a trypsin digestion.Alternatively, phage selected on agonist-bound antigen is eluted usingan excess of antagonist or vice versa.

After two rounds of bio-panning, 180 colonies were randomly picked andthe nanobodies were produced as a soluble His-EPEA-tagged protein in theperiplasm of the TG1 cells. A comparative solid-phase ELISA on M3R-T4Lreceptor versus AT1aR-T4L receptor resulted in 66 M3R-specificnanobodies.

Nanobody Purification for Biochemical Characterization

HIS-tagged or HIS-EPEA-tagged nanobodies were expressed in WK6 E. colicells. Periplasmic extracts were subjected to immobilized metal affinitychromatography on nickel (II) sulfate fast-flow sepharose (GEHealthcare). IMAC protein fractions were dialyzed overnight in 100 mMIVIES pH 6.5, 100 mM NaCl buffer. Dialyzed nanobodies were furtherpurified by cation exchange chromatography (AKTA FPLC with Mono S 10/100GL column).

Size Exclusion Chromatography

Agonist selective nanobodies were identified by size exclusionchromatography following incubation of 20 μM agonist or inverse agonist-(carazolol-) bound β₂AR-365N for one hour at RT in the absence orpresence of 40 μM nanobody. Chromatography was performed in 0.1% DDM, 20mM HEPES pH7.5, 100 mM NaCl in the presence of 1 μM of the respectiveligands using an ÄKTA FPLC with Superdex 200 10/300 GL column.

Ligand Binding on the Truncated β₂AR Receptor in Membrane Preparationsfrom Insect Cells

Competition binding experiments were performed on β₂AR-365 expressed inSf9 insect cell membranes in the absence or presence of 1 μM nanobodyfor 90 minutes at RT in binding buffer (75 mM Tris pH7.5, 12.5 mM MgCl₂,1 mM EDTA, 0.05% BSA, and 10 μM GTPγS) containing 0.5 nM[³H]-dihydroalprenolol and (-)-isoproterenol at concentrations rangingfrom 10⁻¹¹ M to 10⁻⁴ M. Bound radioligand was separated from unboundover Whatman GF/B filters using a Brandel harvester. The data are themean±S.E. of two independent experiments performed in triplicate.

Bimane Fluorescence

Purified β₂AR was reacted with 1:1 equivalent of monobromobimane (mBBr,Invitrogen) in 100 mM NaCl, 20 mM HEPES, pH 7.5, 0.1% dodecyl maltosideand incubated overnight on ice in the dark. The fluorophore-labeledreceptor was purified right before use by gel filtration on a desaltingcolumn equilibrated with the same buffer. Fluorescence spectroscopyexperiments were performed on a Spex FluoroMax-3 spectrofluorometer(Jobin Yvon Inc, NJ) with photon counting mode by using an excitationand emission bandpass of 4 nm. All experiments were performed at 25° C.For emission scans, excitation was set at 370 nm and emission wasmeasured from 430-530 nm with an integration time of 1 s/nm. Todetermine the effect of nanobodies and ligands, three individual labeledprotein samples were incubated with 1 μM nanobody or 10 μM Isoproterenolor both. Emission spectra of the samples were taken after 1 hourincubation. Fluorescence intensity was corrected for backgroundfluorescence from buffer and ligands in all experiments. The data arethe mean±S.E. of two independent experiments performed in triplicate.

Preparation of β₂AR-T4L and Nanobody-80 for Crystallography

β₂AR-T4L was expressed in Sf-9 insect cell cultures infected withβ₂AR-T4L baculovirus, and solubilized according to previously describedmethods (Kobilka 1995). Functional protein was obtained by M1 FLAGaffinity chromatography (Sigma) prior to and followingalprenolol-Sepharose chromatography (Kobilka 1995). In the second M1chromatography step, receptor-bound alprenolol was exchanged for a highaffinity agonist and dodecylmaltoside was exchanged for the MNG-3amphiphile for increased receptor stability (Chae and Gellman,unpublished). The agonist-bound and detergent-exchanged β₂AR-T4L waseluted in 10 mM HEPES pH 7.5, 100 mM NaCl, 0.02% MNG-3, and 10 μMagonist followed by removal of N-linked glycosylation by treatment withPNGaseF (NEB). The protein was concentrated to ˜50 mg/ml with a 100 kDamolecular weight cut off Vivaspin concentrator (Vivascience).

Nanobody-80 (Nb80) bearing a C-terminal His6 tag was expressed in theperiplasm of E. coli strain WK6 following induction with IPGT. Culturesof 0.6 L were grown to OD₆₀₀=0.7 at 37° C. in TB media containing 0.1%glucose, 2 mM MgCl₂, and 50 μg/ml ampicillin. Induced cultures weregrown overnight at 28° C. Cells were harvested by centrifugation andlysed in ice-cold buffer (50 mM Tris pH 8.0, 12.5 mM EDTA, and 0.125 Msucrose), then centrifuged to remove cell debris. Nb80 was purified bynickel affinity chromatography, dialyzed against buffer (10 mM HEPES pH7.5, 100 mM NaCl), and spin concentrated to ˜120 mg/ml.

Crystallization

Agonist-bound β₂AR-T4L and nanobody (e.g., Nb80) were mixed in 1:1.2molar ratio, incubated two hours at RT before mixing with liquefiedmonoolein (M7765, Sigma) containing 10% cholesterol (C8667, Sigma) in1:1.5 protein to lipid ratio (w/w) using the twin-syringe mixing methoddeveloped by Martin Caffrey (Caffrey and Cherezov 2009). Initialcrystallization leads were identified using in-house screens andoptimized in 24-well glass sandwich plates using 50 nL protein:lipiddrops manually delivered and overlaid with 0.8 μl precipitant solutionin each well and sealed with a glass cover slip. Crystals for datacollection were grown at 20° C. by hanging drop vapor diffusion using0.8 μl reservoir solution (36 to 44% PEG 400, 100 mM Tris pH 8.0, 4%DMSO, 1% 1,2,3-heptanetriol) diluted two- to four-fold in Milli-Q water.Crystals grew to full size within seven to ten days. Crystals were flashfrozen and stored in liquid nitrogen with reservoir solution ascryoprotectant.

Microcrystallography Data Collection and Processing

Diffraction data were measured at beamline 23-ID of the Advanced PhotonSource, using a 10 μm diameter beam. Low dose 1.0° rotation images wereused to locate and center crystals for data collection. Data weremeasured in 1.0° frames with exposure times typically five to tenseconds with a 5× attenuated beam. Only 5-10° of data could be measuredbefore significant radiation damage occurred. Data were integrated andscaled with the HKL2000 package (Otwinowski 1997).

Structure Solution and Refinement

Molecular replacement phases were obtained with the program Phaser(McCoy 2007). The search models were 1) the high-resolutioncarazolol-bound β₂AR structure, PDB id 2RH1, but with T4L and all water,ligand and lipid molecules removed) and a nanobody (PDB id 3DWT, watermolecules removed) as search models. The rotation and translationfunction Z scores were 8.7 and 9.0 after placing the β₂AR model, and thenanobody model placed subsequently had rotation and translation functionZ scores of 3.5 and 11.5. The model was refined in Phenix (Afonine 2005)and Buster (Blanc 2004), using a group B factor model with one B formain chain and one B for side chain atoms. Refinement statistics aregiven in Table 5. Despite the strong anisotropy (Table 5), the electrondensity was clear for the placement of side chains.

Ligand Binding on the Truncated β₂AR Receptor Reconstituted in HDLParticles.

The effect of Nb80 and Gs on the receptors affinity for agonists wascompared in competition binding experiments. The β₂AR and β₂AR-T4L (bothtruncated at position 365) purified as previously described (Rosenbaumet al. 2007; Rasmussen et al. 2007) were reconstituted in high-densitylipoprotein (HDL) particles followed by reconstitution of Gs into HDLparticles containing β₂AR according to previously published methods(Whorton et al. 2007). 0.6 nM [³H]-dihydroalprenolol (³H-DHA) was usedas radioligand and (-)-isoproterenol (ISO) at concentrations rangingfrom 10⁴² to 10⁴ M as competitor. Nb80 was used at 1 μM. GTPγS was usedat 10 μM. TBS (50 mM Tris pH 7.4, 150 mM NaCl) containing 0.1% BSA wasused as binding buffer. Bound ³H-DHA was separated from unbound on aBrandel harvester by passing over a Whatman GF/B filter (presoaked inTBS with 0.3% polyethylenimine) and washed in cold TBS. Radioligandbinding was measured in a Beckman LS6000 scintillation counter. Ligandbinding affinity (K_(d)) of DHA was determined from saturation bindingcurves using GraphPad Prism software. Binding affinities of ISO (K_(i)values, tabulated in Table 4) were determined from IC50 values using theequation K_(i)=IC₅₀/(1+[L]/K_(d)).

Ligand Binding on the Full-Length β₂AR Receptor in Membrane Extractsfrom Insect Cells for Improved Agonist Identification

Competition radioligand binding experiments on membrane extracts wereperformed essentially as described by Seifert and co-workers (Seifert etal. 1998. Eur. J. Biochem. 255:369-382). Ten μg of homogenized membraneextracts from insect cells containing human β₂AR (Perkin Elmer, cat nr6110106400UA) were incubated with Nb80 or a non-related Nanobody(negative control; Irr Nb) for one hour at 37° C. in incubation buffer(75 mM Tris-HCl, 12.5 mM MgCl₂, 1 mM EDTA and 0.2% w/v BSA) in 24-wellplates (Corning Costar).

Nanobodies were applied at a final concentration of 500 nM,corresponding to a 3000-fold excess of nanobody versus the adrenergicreceptor. Subsequently, an appropriate dilution series of the ligandunder investigation was added to the nanobody-bound membrane extractstogether with 2 nM of ³H-DHA radioligand (Perkin Elmer cat nrNET720001MC; specific activity of 104.4 Ci/mmol). The total volume perwell was adjusted with incubation buffer to 500 μl and the reactionmixture was further incubated for another hour at 37° C. in a waterbath. After harvesting the membrane extracts with a cell harvester(Inotech) onto glass fiber filters (Whatmann GF/B filter paper), filterswere washed with ice cold wash buffer (50 mM Tris-HCl pH 7.4) and airdried filters parts were transferred to scintillation tubes containing3.5 ml of Optiphase “Hisafe 2” scintillation liquid (Perkin Elmer).Radioactivity was measured in a LKB Wallace scintillation counter afterone hour incubation at room temperature.

Compound Library Screening

A compound library was screened for agonists using a competitionradioligand binding assay. For this purpose, 10 μg of in house preparedmembrane extracts from HEK293T cells expressing human β₂AR (expressionlevel of ˜10 pmol/mg membrane protein) are pre-incubated with Nb80 or anon-related Nanobody (negative control) for one hour at 30° C. inincubation buffer (50 mM Hepes pH 7.4, 1 mM CaCl₂), 5 mM MgCl₂, 100 mMNaCl and 0.5% w/v BSA). Nanobodies are applied at a final concentrationof 500 nM, roughly corresponding to a 3000-fold excess of Nanobodyversus the β₂AR. Subsequently, the Nanobody-loaded membranes are addedto 96-well plates containing library compounds and 2 nM of³H-dihydroalprenolol (DHA) radioligand. The total volume per well isadjusted with incubation buffer to 100 μl and the reaction mixture isfurther incubated for another hour at 30° C. Subsequently,membrane-bound radioligand is harvested using a GF/B glass fiber 96-wellfilterplate (Perkin Elmer) presoaked in 0.3% polyethylenimine. Filterplates are washed with ice-cold wash buffer (50 mM Tris-HCl pH7.4), anddried for 30 minutes at 50° C. After adding 25 μl of scintillation fluid(MicroScint™-O, Perkin Elmer), radioactivity (cpm) is measured in aWallace MicroBeta TriLux scintillation counter.

Bimane Fluorescence Spectroscopy on β₂AR Reconstituted in HDL Particles

To compare the effects on receptor conformation of Gs and Nb80 bindingthe purified β₂AR was labeled with the environmentally sensitivefluorescent probe monobromo-bimane (Invitrogen) at cysteine 265 locatedin the cytoplasmic end of TM6, and reconstituted into HDL particles(mBB-β₂AR/HDL). Prior to obtaining fluorescence emission spectra 10 nMmBB-β₂AR/HDL incubated 30 minutes at RT in buffer (20 mM HEPES pH 7.5,100 mM NaCl) in the absence or presence of 10 μM ISO, 1 μM inverseagonist ICI-118,551 (ICI), 300 nM Gs heterotrimer, or 300 nM Nb80, or incombinations of ISO with Gs, ISO with Nb80, and ICI with Nb80.Fluorescence spectroscopy was performed on a Spex FluoroMax-3spectrofluorometer (Jobin Yvon Inc.) with photon-counting mode, using anexcitation and emission bandpass of 5 nm. Excitation was set at 370 nmand emission was collected from 415 to 535 nm in 1 nm increments with0.3 sec/nm integration time. Fluorescence intensity was corrected forbackground fluorescence from buffer and ligands.

TABLE 1 List of β₂AR-specific nanobodies Nanobody reference numberNanobody short notation SEQ ID NO: CA2764 NB64 1 CA3431 NB31 2 CA3413NB13 3 CA2780 NB80 4 CA2765 NB65 5 CA2761 NB61 6 CA3475 NB75 7 CA2770NB70 8 CA3472 NB72 9 CA3420 NB20 10 CA3433 NB33 11 CA3434 NB34 12 CA3484NB84 13 CA2760 NB60 14 CA2773 NB73 15 CA3477 NB77 16 CA2774 NB74 17CA2768 NB68 18 CA3424 NB24 19 CA2767 NB67 20 CA2786 NB86 21 CA3422 NB2222 CA2763 NB63 23 CA2772 NB72 24 CA2771 NB71 25 CA2769 NB69 26 CA2782NB82 27 CA2783 NB83 28 CA2784 NB84 29

TABLE 2 CDRs of β₂AR-specific nanobodies Nanobody Nanobody referenceshort number notation CDR1 CDR2 CDR3 CA2764 NB64 GSIFSINT IHSGGSTNVKDYGAVLYEYDY (SEQ ID NO: 30) (SEQ ID NO: 43) (SEQ ID NO: 57) CA3431NB31 GSIFSINT IHSGGST NVKDYGAVLYEYDY (SEQ ID NO: 30) (SEQ ID NO: 43)(SEQ ID NO: 57) CA3413 NB13 GSIFSINT IHSGGST NVKDYGAVLYEYDY(SEQ ID NO: 30) (SEQ ID NO: 43) (SEQ ID NO: 57) CA2780 NB80 GSIFSINTIHSGGST NVKDYGAVLYEYDY (SEQ ID NO: 30) (SEQ ID NO: 43) (SEQ ID NO: 57)CA2765 NB65 GSIFSINT IHSGGST NVKDYGAVLYEYDY (SEQ ID NO: 30)(SEQ ID NO: 43) (SEQ ID NO: 57) CA2761 NB61 GSIFSLND ITSGGSTNAVVAGTFSTYDY (SEQ ID NO: 31) (SEQ ID NO: 44) (SEQ ID NO: 58) CA3475NB75 GSIFSLND ITSGGST NAVVAGTFSTYDY (SEQ ID NO: 31) (SEQ ID NO: 44)(SEQ ID NO: 58) CA2770 NB70 GSIFSLND ISSGGRL NAVVAGTFSTYDY(SEQ ID NO: 31) (SEQ ID NO: 45) (SEQ ID NO: 58) CA3472 NB72 GSIFSLNDISSGGRL NAVVAGTFSTYDY (SEQ ID NO: 31) (SEQ ID NO: 45) (SEQ ID NO: 58)CA3420 NB20 GSIFSLND ITSGGST NAKVAGTFSIYDY (SEQ ID NO: 31)(SEQ ID NO: 44) (SEQ ID NO: 59) CA3433 NB33 GSIFSLND VTSGGSTNAKVAGTFSIYDY (SEQ ID NO: 31) (SEQ ID NO: 46) (SEQ ID NO: 59) CA3434NB34 GSIFSLND ITSGGST NAKVAGTFSIYDY (SEQ ID NO: 31) (SEQ ID NO: 44)(SEQ ID NO: 59) CA3484 NB84 GSIFSLND ITSGGST NAKVAGTFSIYDY(SEQ ID NO: 31) (SEQ ID NO: 44) (SEQ ID NO: 59) CA2760 NB60 GSIFSLNDITSGGST NAKVAGTFSIYDY (SEQ ID NO: 31) (SEQ ID NO: 44) (SEQ ID NO: 59)CA2773 NB73 GSIFSLND ITSGRST NAKVAGTFSIYDY (SEQ ID NO: 31)(SEQ ID NO: 47) (SEQ ID NO: 59) CA3477 NB77 GSIFSLND ITSGGSTNAKVAGTFSIYDY (SEQ ID NO: 31) (SEQ ID NO: 44) (SEQ ID NO: 59) CA2774NB74 GSIFSIND ITSGGSV NAKVAGTFSIYDY (SEQ ID NO: 32) (SEQ ID NO: 48)(SEQ ID NO: 59) CA2768 NB68 GTIFSNNA ITSGGST NAKVPGTFSIYDY(SEQ ID NO: 33) (SEQ ID NO: 44) (SEQ ID NO: 60) CA3424 NB24 GSVFSLPTITGSGST YYRSTFTEY (SEQ ID NO: 34) (SEQ ID NO: 49) (SEQ ID NO: 61) CA2767NB67 GTISSFIA ITSGGET NAQVFADIFNLINY (SEQ ID NO: 35) (SEQ ID NO: 50)(SEQ ID NO: 62) CA2786 NB86 GTIFSPNT ITSGGSR NYQTVFFGNAEA(SEQ ID NO: 36) (SEQ ID NO: 51) (SEQ ID NO: 63) CA3422 NB22 GSIFSINASTSGDIT NARGIYSDYAFADFNS (SEQ ID NO: 37) (SEQ ID NO: 52) (SEQ ID NO: 64)CA2763 NB63 GSRFSFIT LSGDNT (SEQ RGTSVLYDV (SEQ ID NO: 38) ID NO: 53)(SEQ ID NO: 65) CA2772 NB72 GFTFSGYA INSGGGST (SEQ HARDIYSDFLGQYEYDY(SEQ ID NO: 39) ID NO: 54) (SEQ ID NO: 66) CA2771 NB71 GFAFSSYE ITTGGNTNANWDLLSDY (SEQ ID NO: 40) (SEQ ID NO: 55) (SEQ ID NO: 67) CA2769 NB69GSIFSINA ITSGGST NVQGTGPSSWLFNEYDY (SEQ ID NO: 37) (SEQ ID NO: 44)(SEQ ID NO: 68) CA2782 NB82 GSIFSINS ITSDGST NADSVYSDFLGKYEYDY(SEQ ID NO: 41) (SEQ ID NO: 56) (SEQ ID NO: 69) CA2783 NB83 GSIFSLNAITSDGST NADSVYSDFLGKYEYDY (SEQ ID NO: 42) (SEQ ID NO: 56)(SEQ ID NO: 69) CA2784 NB84 GSIFSINA ITSGGST HVRDIYSDFLGQYEYDY(SEQ ID NO: 37) (SEQ ID NO: 44) (SEQ ID NO: 70)

TABLE 3. Full Agonist Binding Properties of Membranes Expressing β₂AR inthe Presence and Absence of Nanobodies.

[3H]-DHA Competition binding was performed on Sf9 insect cell membranesexpressing β₂AR, in the presence or absence of 1 μM nanobodies. Datarepresent the mean±s.e. of two independent experiments performed intriplicate. The IC50 values used for calculations of Ki values wereobtained from means of pIC50 values determined by nonlinear regressionanalysis using Prism (GraphPad Software, San Diego, Calif.) and the s.e.interval from pIC50±s.e.

TABLE 3 Full agonist binding properties of membranes expressing β₂AR inthe presence and absence of nanobodies. Isoproterenol Ki [S.E. interval](nm) β₂AR  295 [211-412] +NB65 13.8 [6.98-27.3] +NB67 19.4 [11.3-33.1]+NB69 53.5 [34.3-83.1] +NB71 10.6 [3.68-30.4] +NB72  145 [93.3-226]+NB80 10.0 [4.74-21.0] +NB84 75.4 [35.9-158]

[3H]-DHA Competition binding was performed on Sf9 insect cell membranesexpressing β₂AR, in the presence or absence of 1 μM nanobodies. Datarepresent the mean±s.e. of two independent experiments performed intriplicate. The IC50 values used for calculations of Ki values wereobtained from means of pIC50 values determined by nonlinear regressionanalysis using Prism (GraphPad Software, San Diego, Calif.) and the s.e.interval from pIC50±s.e.

TABLE 4 Pharmacological characterization of β₂AR reconstituted in HDLparticles in complex with Gs and Nb80 [³H]-DHA/(−)-isoproterenol[³H]-DHA competition binding saturation binding Low affinity state Highaffinity state K_(d) ± S.E. K_(i) [S.E. interval] K_(i) [S.E. interval]nM nM nM β₂AR 0.55 ± 0.09 (n = 3) 107.5 [103.8-111.3] (n = 3) β₂AR + Gs95.3 [82.8-109.7] 1.07 [0.96-1.19] (n = 4) β₂AR + Gs + GTPγS 95.2[92.2-98.3] (n = 3) β₂AR + NB80 1.13 [1.09-1.18] (n = 3) β₂AR-T4L 0.42 ±0.01 (n = 3) 33.5 [31.6-35.5] (n = 3) β₂AR-T4L + NB80 0.56 [0.54-0.57](n = 4)

TABLE 5 X-ray data collection and refinement statistics of the β₂AR-Nb80complex A. Data collection statistics wavelength (Å)  1.0332 space groupC2 unit cell parameters a (Å) 236.7 b (Å) 45.7 c (Å) 71.4 β (°) 102.3number of crystals 23 resolution (Å) 37-3.50 (3.56-3.50) uniquereflections 10147 (903) completeness 94.8 (93.7) multiplicity 3.5 (3.2)<I/σ(I)> 6.7 (1.8) R_(merge) (%) 0.192 (0.594) B. refinement statisticsresolution (Å) 37-3.50 No. of reflections 9210 (937) working set (testset) R_(work)/R_(free) (%) 0.225/0.294 rmsd from ideality bond lengths(Å) 0.010 bond angles (°) 1.3 Anisotropic B B₁₁ = 33.5/B₂₂ = 2.7/B₃₃ =−36.3/B₁₃ = 4.5 correction (Å²) Average B factor (Å²) receptor 76.4nanobody 96.6 agonist 62.4 Ramachandran analysis residues inmost-favored 86.6 region (%) additionally allowed 13.4 region (%)generously allowed 0.0 region (%) disallowed region (%) 0.0

TABLE 6 Nanobody 80 induced potency shift of (ant-)agonists. Agonistsshow an increased affinity for the β₂AR-Nb80 complex. Type competitorfor Competitor ID β₂AR Potency shift* isoproterenol agonist 8.8alprenolol antagonist 1.1 carvedilol antagonist 1.2 CGP12177A antagonist1.0 salmeterol full agonist 3.3 terbutaline partial agonist 4.3dobutamine partial agonist 2.3 salbutamol partial agonist 4.2 ICI118,551Inverse agonist 0.9 metaproterenol agonist 2.9 procaterol agonist 2.3ritodrine agonist 2.7 fenoterol Full agonist 3.7 formoterol agonist 4.0timolol antagonist 0.6 *Potency shifts were determined as the ratio ofthe IC50 measured in the presence of an irrelevant Nb (negative control)and the IC50 measured in the presence of Nb80.

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1.-42. (canceled)
 43. A host cell, wherein the host cell expresses anexpression vector encoding a nanobody, or a functional fragment thereof,and wherein the nanobody specifically binds to an intracellularconformational epitope of a GPCR.
 44. The host cell of claim 1, whereinthe nanobody or functional fragment thereof stabilizes or induces afunctional conformational state of the GPCR upon binding of the nanobodyor functional fragment thereof to the GPCR.
 45. The host cell of claim1, wherein the nanobody or functional fragment thereof specificallybinds a conformational epitope of the GPCR that is comprised in abinding site for a downstream signaling protein.
 46. The host cell ofclaim 1, wherein the nanobody or functional fragment thereofspecifically binds a conformational epitope of the GPCR that iscomprised in, located at, or overlaps with the G protein binding site ofthe GPCR.
 47. The host cell of claim 1, wherein the nanobody orfunctional fragment thereof occupies the G protein binding site of afunctional conformational state of the GPCR, when bound to the GPCR. 48.The host cell of claim 1, wherein the nanobody or functional fragmentthereof occupies the G protein binding site of an active conformationalstate of the GPCR, when bound to the GPCR.
 49. The host cell of claim 1,wherein the nanobody or functional fragment thereof preferentially bindsagonist-bound receptor versus inverse agonist-bound receptor.
 50. Thehost cell of claim 1, wherein the nanobody or functional fragmentthereof specifically binds to the GPCR when it bound to an agonist. 51.The host cell of claim 1, wherein the nanobody or functional equivalentthereof enhances the affinity of the GPCR for an agonist.
 52. The hostcell of claim 1, wherein the nanobody or functional equivalent thereofenhances the affinity of a GPCR for an agonist at least twofold uponbinding to the GPCR.
 53. The host cell of claim 1, wherein the nanobodyor functional equivalent thereof captures the GPCR in a functionalconformational state.
 54. The host cell of claim 1, wherein the nanobodyor functional equivalent thereof captures the GPCR in an activeconformational state.
 55. The host cell of claim 1, wherein the nanobodyor functional equivalent thereof captures the GPCR bound to a receptorligand in a functional conformational state.
 56. The host cell of claim1, wherein the nanobody or functional equivalent thereof captures theGPCR bound to a receptor ligand in an active conformational state.
 57. Amethod of capturing a GPCR in a functional conformational state, themethod comprising: contacting the GPCR with the host cell of claim 1;and capturing the GPCR with the nanobody or functional fragment thereof.58. A method of capturing a GPCR in an active conformational state, themethod comprising: contacting the GPCR with the host cell of claim 1;and capturing the GPCR with the nanobody or functional fragment thereof.59. A method of capturing a GPCR bound to receptor ligand in afunctional conformational state, the method comprising: contacting theGPCR with the host cell of claim 1; and capturing the GPCR with thenanobody or functional fragment thereof.
 60. A method of capturing aGPCR bound to receptor ligand in an active conformational state, themethod comprising: contacting the GPCR with the host cell of claim 1;and capturing the GPCR with the nanobody or functional fragment thereof.61. The host cell of claim 1, wherein the nanobody or a functionalequivalent thereof enhances the affinity of a GPCR for an agonist atleast fivefold, upon binding to the GPCR.
 62. The host cell of claim 1,wherein the nanobody or a functional equivalent thereof enhances theaffinity of a GPCR for an agonist at least tenfold, upon binding to theGPCR.